Methods and composition for producing and using immune cells and stem cells for cell-based therapies

ABSTRACT

Described herein are methods for selecting lymphocytes for adoptive cell therapy based on P-glycoprotein expression and compositions comprising same.

CROSS REFERENCE TO RELATED APPLICATIONS

The application is a continuation of U.S. application Ser. No.16/315,153, filed Jan. 3, 2019, which application is a U.S. NationalStage Application filed under 35 U.S.C. § 371 and claims priority toInternational Application No. PCT/US2017/042248, filed Jul. 14, 2017,which application claims priority under 35 U.S.C. § 119 to U.S.Provisional Application Ser. No. 62/362,497, filed Jul. 14, 2016, thedisclosures of which are incorporated herein by reference.

TECHNICAL FIELD

Provided herein are methods for selecting lymphocytes for adoptive celltherapy based and methods of using such lymphocytes.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application includes a sequence listing entitled,“00130-010US2.xml” created on Aug. 2, 2022 and having 11,692 bytes ofdata, machine formatted on IBM-PC, MS-Windows operating system usingWIPO Standard ST.26 formatting. The sequence listing is herebyincorporated by reference in its entirety for all purposes.

BACKGROUND

Immunotherapy using adoptive transfer of tumor-specific T cells andchimeric antigen receptor (CAR) and T cell receptor (TCR) modified Tcells mediates durable and complete disease regression in some patientswith metastatic cancer. Recent studies have suggested that metabolismsupports and drives many basic features of T cells, including cellularactivation, proliferation, differentiation, effector function, andantitumor immunity. This has led to a growing interest in leveragingthis understanding to improve the efficacy of T cell transfer therapies,such as adoptive transfer immunotherapy in the treatment of cancer.

Although there is increasing evidence that metabolism can affect thesurvival and antitumor function of T cells, identifying a simple andclinically feasible method to isolate T cells with favorable metabolicfeatures has proved challenging. Because mitochondria are the centralmetabolic organelle in cells, Sukumar et al. (Cell Metabolism, 23:63-76,2016) hypothesized that the measurement of a singlemitochondrial-associated parameter may help to identify T cells with afavorable bioenergetic profile that can survive in vivo for long periodsafter adoptive transfer for T cell-based immunotherapy, such as CAR-T,TCR and chimeric T cell receptor (cTCR) based therapies.

Sukumar et al. described a clinically feasible method to isolatefunctionally robust T cells based on a single metabolic parameter:mitochondrial membrane potential (ΔΨm). Mitochondria produce energy byestablishing an electrochemical proton motive force (Δp) across theirinner cell membrane, which in turn fuels the synthesis of ATP by drivingthe proton turbine F0F1 ATPase. Sukumar et al. utilized a lipophiliccationic dye tetramethylrhodamine methyl ester (TMRM) to identify andisolate metabolically robust T cells based on their mitochondrialmembrane potential (ΔΨm). They showed that CD8+ T cells that are foundto have low-ΔΨm display enhanced in vivo persistence and greaterantitumor immunity relative to high-ΔΨm cells. Based on these findings,the authors claimed that they have demonstrated that metabolic sortingcan complement sorting based on conventional cell surface markers inidentifying cells with the capacity for long-term survival and ongoingeffector function after adoptive transfer. They further believed thatimmunometabolomic approach to cell sorting may have important andimmediate therapeutic applications in enhancing cell-based therapies forpatients with viral-associated illness, advanced cancer, and disordersof hematopoiesis. However, sorting of lymphocytes based on TMRM stainingand flow sorting is slow and expensive and therefore not readilyamenable to clinical application.

SUMMARY

The disclosure provides a method for isolating cells suitable foradoptive cell therapy. The method comprises isolating p-glycoprotein(Pgp) positive cells. The disclosure provides a method for isolatingcells suitable for adoptive cell therapy. The method comprises obtaininga sample; optionally enriching the sample for T cells, NK cells, stemcells, and/or mononuclear cells; and isolating p-glycoprotein positive(Pgp-positive) including cells selected from the group consisting of Tcells, NK cells, stem cells, and/or mononuclear cells from the enrichedsample, so as to obtain a fraction enriched in Pgp-positive cells,thereby isolating cells suitable for adoptive cell transfer therapy. Ina further embodiment, the method comprises contacting the sample with atleast one cytotoxic drug that is a substrate of Pgp at a concentrationappropriate to kill Pgp-negative T cells, NK cells and/or differentiatedcells. In further embodiment, the at least one cytotoxic drug is any oneor more of vincristine, vinblastin, doxorubicin, daunorubicin, taxol,paclitaxol, etoposide, mitoxantrone, actinomycin-D, or combinationsthereof. In still a further embodiment or alternative embodiment, themethod comprises contacting the sample with at least one phototoxiccompound; and exposing the sample to a visible light source sufficientto activate the at least one phototoxic compound so as to killPgp-negative T cells. In a further embodiment, the at least onephototoxic compound is any one or more of2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid methylester hydrochloride,2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid ethyl esterhydrochloride, 2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoicacid octyl ester hydrochloride,2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid n-butylester hydrochloride, 2-(6-ethyl amino-3-ethylimino-3H-xanthen-9-yl)-benzoic acid n-butyl ester hydrochloride, orderivatives thereof or combinations thereof. In yet another or furtherembodiment, the method comprises exposing the sample to a physicalstress, such as hyperthermic conditions, that selectively killsPgp-negative cells. In yet another or further embodiment, the methodcomprises exposing the sample to a nutritional or metabolic stress, suchas serum starvation or growth factor starvation, which selectively killspgp-negative cells. In still another or alternative embodiment of any ofthe foregoing the method includes isolating the Pgp-positive cells fromthe sample by exposing the sample to at least one primary antibody orantibody-like moiety specific to p-glycoprotein. In a furtherembodiment, the at least one primary antibody or antibody like moiety isconjugated to at least one fluorescent label or at least one magneticlabel or biotin. In still a further embodiment, the method includesoptionally staining the sample with at least one secondary antibody. Ina further embodiment, the at least one secondary antibody is conjugatedto at least one fluorescent label or at least one magnetic label orbiotin. In still another embodiment of any of the foregoing, theisolating of the Pgp-positive cells from the sample is performed by anyone or more methods selected from immunofluorescent methods,immunomagnetic methods, immunoaffinity methods, or combinations thereof.In still another embodiment of any of the foregoing, the isolating ofthe Pgp-positive cells from the sample is performed by any one or moremethods selected from flow cytometry, magnetic activated cell sorting,biotin-streptavidin affinity purification, or combinations thereof. Instill another embodiment of any of the foregoing, the fraction enrichedin Pgp-positive cells contains less than 50%, less than 40%, less than30%, less than 20%, less than 10%, less than 5%, or less than 1%Pgp-negative cells. In yet a further embodiment, the disclosure providestha the Pgp-positive cells are genetically modified. In yet a furtherembodiment, the disclosure provides that the Pgp-positive T cells, NKcells, stem cells, and/or mononuclear cells are further geneticallymodified to express at least one chimeric antigen receptor, T cellreceptor, chimeric T cell receptor, synthetic immune receptor, TRuC™ orArtemis™ T cell platform, so as to obtain genetically modifiedPgp-positive T cells, NK cells, stem cells, and/or mononuclear cells.

The disclosure also provides pharmaceutical compositions comprising thePgp-positive T cells and/or NK cells and/or stem cells of the disclosureand at least one pharmaceutically acceptable carrier. The disclosurealso provides pharmaceutical compositions comprising the geneticallymodified Pgp-positive T cells and/or NK cells and/or stem cells and atleast one pharmaceutically acceptable carrier. The pharmaceuticalcompositions can be administered to a human or other subject to treatcancer, immune disorders, or infections.

The disclosure also provides a method for treating cancer, immune orinfectious disorders in a subject, comprising administering atherapeutically effective amount of a composition of the disclosurecomprising a Pgp-positive T cells and/or NK cells or geneticallyengineered population thereof to the subject so as to treat cancer,immune or infectious disorders. In one embodiment, the cancer is B-celllymphomas, T cell lymphomas, myeloma, myelodysplastic syndrome, skincancer, brain tumor, breast cancer, colon cancer, rectal cancer,esophageal cancer, anal cancer, cancer of unknown primary site,endocrine cancer, testicular cancer, lung cancer, hepatocellular cancer,gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer,liver cancer, bladder cancer, cancer of the urinary tract, cancer ofreproductive organs, thyroid cancer, renal cancer, carcinoma, melanoma,head and neck cancer, brain cancer, prostate cancer, or leukemia. Inanother embodiment, the method further comprises administering achemotherapeutic agent. In still another embodiment, thechemotherapeutic agent is selected from alkylating agents, alkylsulfonates, aziridines, ethylenimines, methylamelamines, acetogenins, acamptothecin, bryostatin, callystatin, CC-1065, cryptophycins,dolastatin, duocarmycin, eleutherobin, pancratistatin, a sarcodictyin,spongistatin, nitrogen mustards, nitrosureas, antibiotics, dynemicin,bisphosphonates, an esperamicin, neocarzinostatin chromophore andrelated chromoprotein enediyne antibiotic chromophores, aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin, deoxydoxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, anti-metabolites, folic acidanalogues, purine analogs, pyrimidine analogs, androgens, anti-adrenals,folic acid replenisher, aceglatone, aldophosphamide glycoside,aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene,edatraxate, defofamine, demecolcine, diaziquone, elformithine,elliptinium acetate, an epothilone, etoglucid, gallium nitrate,hydroxyurea, lentinan, lonidainine, maytansinoids, mitoguazone,mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet,pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide,procarbazine, razoxane, rhizoxin, sizofuran, spirogermanium, tenuazonicacid, triaziquone, 2,2′,2″-trichlorotriethylamine, trichothecenes,urethane, vindesine, dacarbazine, mannomustine, mitobronitol,mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide,thiotepa, taxoids, chloranbucil, 6-thioguanine, mercaptopurine,methotrexate, platinum analogs, vinblastine, platinum, etoposide(VP-16), ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone,teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate,irinotecan, topoisomerase inhibitor RFS 2000; difluoromethylornithine,retinoids, capecitabine, combretastatin, leucovorin, oxaliplatin,lapatinib, inhibitors of PKC-alpha, Raf, H-Ras, EGFR and VEGF-A,tyrosine kinase inhibitors, nucleoside analogs, mTOR inhibitors,Bcl2-family inhibitors, immunomodulatory drugs and proteasome inhibitorsthat reduce cell proliferation, and pharmaceutically acceptable salts,acids or derivatives of any of the above, or combinations thereof. In afurther embodiment, the composition and the chemotherapeutic agent areadministered sequentially or simultaneously. In another embodiment, themethod further comprises administering an anticancer agent, such as anantibody, antibody drug conjugate, bispecific antibody, DART, a singledomain antibody, or a non-immunoglobulin antigen binding scaffold. Inanother embodiment, the method further comprises performing stem celltransplant. In another embodiment, the method further comprisesadministering an anti-infective agent. In another embodiment, the methodfurther comprises administering an immunosuppressive agent.

The disclosure also provides a method for isolating Pgp-negative Tcells, NK cells, and/or mononuclear cells suitable for adoptive celltransfer therapy, comprising obtaining a sample; optionally enrichingthe sample for T cells, NK cells, and/or mononuclear cells; anddepleting Pgp-positive cells from the sample, so as to obtain a fractionenriched in Pgp-negative cells suitable for adoptive cell transfertherapy. In one embodiment, the method comprises depleting thePgp-positive cells from the sample by exposing the sample to at leastone primary antibody or antibody like moiety specific to p-glycoprotein.In a further embodiment, the at least one primary antibody or antibodylike moiety is conjugated to at least one fluorescent label or at leastone magnetic label or biotin. The method optionally can include stainingthe sample with at least one secondary antibody. In another embodiment,the at least one secondary antibody is conjugated to at least onefluorescent label or at least one magnetic label or biotin. In stillanother embodiment, the depleting of the Pgp-positive cells from thesample is performed by any one or more methods selected fromimmunofluorescent methods, immunomagnetic methods, or immunoaffinitymethods, or combinations thereof. In yet another embodiment, thedepleting of the Pgp-positive cells from the sample is performed by flowcytometry, magnetic activated cell sorting, or biotin-streptavidin basedcell sorting, or combinations thereof. In a further embodiment of any ofthe foregoing, the fraction enriched in Pgp-negative T cells, NK cells,and/or mononuclear cells contains less than 50%, less than 40%, lessthan 30%, less than 20%, less than 10%, less than 5%, or less than 1%Pgp-positive cells.

The disclosure also provides a pharmaceutical composition, comprising anamount of the fraction enriched in Pgp-negative lymphocytes, T cells orNK cells and at least one pharmaceutically acceptable carrier.

The disclosure also provides a method of treating an infection in animmunodeficient HIV/AIDS subject, comprising administering atherapeutically effective amount of the composition comprising thefraction enriched in Pgp-negative T cells and/or NK cells to the subjectso as to treat the infection. In one embodiment, the infection is causedby a viral, bacterial, fungal or protozoan pathogen. In a furtherembodiment, the infection is caused by cytomegalovirus, adenovirus,adeno-associated virus, BK virus, Human Herpesvirus 6, Human Herpesvirus8, Epstein Barr virus, influenza virus, parainfluenza virus, measlesvirus, mumps virus, rhino virus, varicella virus, herpes simplex virus 1and 2, HIV-1, HTLV1, Mycobacterium tuberculosis, atypical mycobacteriaspecies, toxoplasmosis, nocardia, aspergillus, mucor, or candida.

The disclosure also provides a method for reducing graft-versus-hostdisease in a subject undergoing an allogeneic stem cell transplant,comprising administering a therapeutically effective amount of thecomposition comprising the fraction enriched in Pgp-negative T cellsand/or NK cells to the subject so as to reduce graft-versus-hostdisease.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-L shows immunofluorescence staining and sorting to enrich forPgp expression cells.

FIG. 2 shows a decline in cell viability following exposure to light inboth TH9402-treated compared to non-treated cells.

FIG. 3 shows that serum starvation results in a significant enrichmentof CD34⁺ stem cells.

FIG. 4 shows that growth factor starvation resulted in an enrichment ofCD34⁺ cells.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” includes aplurality of such cells and reference to “the polynucleotide” includesreference to one or more polynucleotides and so forth.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Allen et al., Remington: TheScience and Practice of Pharmacy 22^(nd) ed., Pharmaceutical Press (Sep.15, 2012); Hornyak et al., Introduction to Nanoscience andNanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary ofMicrobiology and Molecular Biology 3^(rd) ed., revised ed., J. Wiley &Sons (New York, N.Y. 2006); Smith, March's Advanced Organic ChemistryReactions, Mechanisms and Structure 7^(th) ed., J. Wiley & Sons (NewYork, N.Y. 2013); Singleton, Dictionary of DNA and Genome Technology3^(rd) ed., Wiley-Blackwell (Nov. 28, 2012); and Green and Sambrook,Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring HarborLaboratory Press (Cold Spring Harbor, N.Y. 2012), provide one skilled inthe art with a general guide to many of the terms used in the presentapplication. For references on how to prepare antibodies, seeGreenfield, Antibodies A Laboratory Manual 2^(nd) ed., Cold SpringHarbor Press (Cold Spring Harbor N.Y., 2013); Köhler and Milstein,Derivation of specific antibody-producing tissue culture and tumor linesby cell fusion, Eur. J. Immunol. 1976 July, 6(7):511-9; Queen andSelick, Humanized immunoglobulins, U.S. Pat. No. 5,585,089 (1996December); and Riechmann et al., Reshaping human antibodies for therapy,Nature 1988 Mar. 24, 332(6162):323-7 All headings and subheadingprovided herein are solely for ease of reading and should not beconstrued to limit the invention. Although methods and materials similaror equivalent to those described herein can be used in the practice ortesting of the invention, suitable methods and materials are describedbelow. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andspecific examples are illustrative only and not intended to be limiting.

As used herein “beneficial results” may include, but are in no waylimited to, lessening or alleviating the severity of the diseasecondition, preventing the disease condition from worsening, curing thedisease condition, preventing the disease condition from developing,lowering the chances of a patient developing the disease condition andprolonging a patient's life or life expectancy.

“Cancer” and “cancerous” refer to or describe the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth. Examples of cancer include, but are not limited to B-celllymphomas (Hodgkin's lymphomas and/or non-Hodgkins lymphomas), T celllymphomas, myeloma, myelodysplastic syndrome, skin cancer, brain tumor,breast cancer, colon cancer, rectal cancer, esophageal cancer, analcancer, cancer of unknown primary site, endocrine cancer, testicularcancer, lung cancer, hepatocellular cancer, gastric cancer, pancreaticcancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer,cancer of the urinary tract, cancer of reproductive organs thyroidcancer, renal cancer, carcinoma, melanoma, head and neck cancer, braincancer (e.g., glioblastoma multiforme), prostate cancer, including butnot limited to androgen-dependent prostate cancer andandrogen-independent prostate cancer, and leukemia. Other cancer andcell proliferative disorders will be readily recognized in the art.

“Chemotherapeutic agents” are compounds that are known to be of use inchemotherapy for cancer. Non-limiting examples of chemotherapeuticagents can include alkylating agents such as thiotepa and CYTOXAN□cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE; vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (Tykerb); inhibitors of PKC-alpha, Raf,H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cellproliferation and pharmaceutically acceptable salts, acids orderivatives of any of the above or combinations thereof.

“Chimeric antigen receptors” (CAR) are artificial T cell receptorstypically used as a therapy for cancer, using a technique calledadoptive cell transfer. The essential antigen-binding, signaling, andstimulatory functions of the TCR complex have been reduced by geneticrecombination methods to a single polypeptide chain, generally referredto as a Chimeric Antigen Receptor (CAR). See, e.g., Eshhar, U.S. Pat.No. 7,741,465; Eshhar, U.S. Patent Application Publication No.2012/0093842. CARs are constructed specifically to stimulate T cellactivation and proliferation in response to a specific antigen to whichthe CAR binds. Typically “CAR-T cells” are used, which refer to T-cellsthat have been engineered to containing a chimeric antigen receptor.Thus, T lymphocytes bearing such CARs are generally referred to as CAR-Tlymphocytes. As described more fully, below, the disclosure provides forthe isolation as well as isolated Pgp-positive cells that can be or aremodified so that they express receptors that recognize proteins that arespecific to the particular form of cancer. Such cells can bereintroduced to a subject, wherein the cells recognize and kill cancercells. CARs have also been used for adoptive cell therapy of immune andinfectious diseases. In addition to CAR, native, engineered and chimericT cell receptors (TCR) have also been used for adoptive cell therapy.

“Disease targeted by genetically modified cells” as used hereinencompasses the targeting of any cell involved in any manner in anydisease by a genetically modified cells the hones to the disease or atarget tissue or cell type, irrespective of whether the geneticallymodified cells target diseased cells or healthy cells to effectuate atherapeutically beneficial result.

“Genetically modified cells”, “redirected cells”, “geneticallyengineered cells” or “modified cells” as used herein refer to cells thathave been modified to express a CAR and native, engineered or chimericTCR. For example, a genetically modified T-lymphocyte that expresses aCAR is a genetically modified cell (sometimes referred to as a CAR-Tcell).

“Mammal” as used herein refers to any member of the class Mammalia,including, without limitation, humans and nonhuman primates such aschimpanzees and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats and guineapigs, and the like. The term does not denote a particular age or sex.Thus, adult and newborn subjects, as well as fetuses, whether male orfemale, are intended to be included within the scope of this term.

“P-glycoprotein” (P-gp or Pgp) is an ATP-dependent efflux pump withbroad substrate specificity that pumps many foreign substances out ofcells. P-glycoprotein is also known as multidrug resistance protein 1(MDR1) or ATP-binding cassette sub-family B member 1 (ABCB1) or clusterof differentiation 243 (CD243). As used herein and as understood by oneof skill in the art, Pgp-positive is abbreviated (Pgp⁺) and Pgp-negativeis abbreviated (Pgp⁻).

“Stem cells” are cells capable of differentiation into other cell typesand/or which retain the ability to continually replicate. Stem cellsinclude those having a particular, specialized function (e.g., tissuespecific cells, parenchymal cells and progenitors thereof). Progenitorcells (i.e., “multipotent”) are cells that can give rise to differentterminally differentiated cell types, and cells that are capable ofgiving rise to various progenitor cells. Cells that give rise to some ormany, but not all, of the cell types of an organism are often termed“pluripotent” stem cells, which are able to differentiate into any celltype in the body of a mature organism. As will be appreciated,“multipotent” stem/progenitor cells have a more narrow differentiationpotential than do pluripotent stem cells. Another class of cells evenmore primitive (i.e., uncommitted to a particular differentiation fate)than pluripotent stem cells are the so-called “totipotent” stem cells(e.g., fertilized oocytes, cells of embryos at the two and four cellstages of development), which have the ability to differentiate into anytype of cell of the particular species. For example, a single totipotentstem cell could give rise to a complete animal, as well as to any of themyriad of cell types found in the particular species (e.g., humans).Moreover, in the case of lymphocytes a “stem cell lymphocyte” or“lymphocyte progenitor” can be a lymphocyte that has not been“activated” to target a particular antigen. Such lymphocyte progenitorsretain the ability to be “activated” to target a particular antigen.Such lymphocyte progenitor cells are particularly useful in generatingCAR-T cells as the progenitors can be readily targeted using arecombinant CAR and have greater replicative potential. Stem cells andprogenitor cells of the disclosure include CD34⁺ cells. The term“pluripotent hematopoietic stem cell” refers to a hematopoietic stemcell that can give rise to all blood cell types.

“Target cell” as used herein refers to cells which are involved in adisease and can be targeted to prevent or treat a disease condition bygenetically modified cells of the disclosure (including but not limitedto genetically modified T-cells, NK cells, hematopoietic stem cells,pluripotent stem cells, and embryonic stem cells). Other target cellswill be apparent to those of skill in the art and may be used inconnection with alternate embodiments of the disclosure.

The terms “T-cell” and “T-lymphocyte” are interchangeable and usedsynonymously herein. Examples include but are not limited to naïve Tcells (“lymphocyte progenitors”), central memory T cells, effectormemory T cells, stem memory T cells (T_(scm)) or combinations thereof.

Pgp positive lymphocytes of the disclosure include the following:

(1) T cells—(a) Pgp⁺ T cells and subsets as defined by one or moreimmunological markers, such as CD8⁺, CD4⁺, CD62L⁺, CCR7⁺ etc.; (b) Pgp⁺T cells can also include different stages of differentiation, such asnaïve T cells (“lymphocyte progenitors”), central memory T cells,effector memory T cells, memory stem T cells (T_(SCM)) etc.; (c) Pgp⁺ Tcells can also include different functional subclasses, such as Helper Tcells, Cytotoxic T cells, Natural Killer T cells or regulatory T cellsetc.; and (d) Pgp⁺ T cells can also be classified based on the site fromwhich they are obtained, such as peripheral blood, lymph nodes, spleen,bone marrow, tissue resident lymphocytes, tumor infiltrating lymphocytesetc.; and

(2) NK Cells (Natural Killer cells)—(a) Pgp⁺ NK cells and subsets asdefined by one or more immunological markers, such as CD56hi, CD56loetc.; (b) Pgp⁺ NK cells can also include NK cells at different stages ofdifferentiation, such as naïve NK cells etc.; (c) Pgp⁺ NK cells can alsobe classified based on the site from which they are obtained, such asperipheral blood, lymph nodes, spleen, bone marrow, tissue residentlymphocytes, tumor infiltrating lymphocytes etc.

Pgp negative lymphocytes (e.g., for use in patients undergoingallogeneic stem cell transplantation to reduce the incidence of GVHD)include the following subsets:

(1) T cells—(a) Pgp⁻ T cells and subsets as defined by one or moreimmunological markers, such as CD8⁺, CD4⁺, CD62L⁺, CCR7⁺ etc.; (b) Pgp⁻T cells can also include T cells at different stages of differentiation,such as naïve T cells (“lymphocyte progenitors”), central memory Tcells, effector memory T cells, memory stem T cells (TSCM) etc.; (c)Pgp⁻ T cells can also include cells belonging to different functionalsubclasses, such as Helper T cells, Cytotoxic T cells, Natural Killer Tcells or regulatory T cells etc.; and (d) Pgp⁻ T cells can also beclassified based on the site from which they are obtained, such asperipheral blood, lymph nodes, spleen, bone marrow, tissue residentlymphocytes, tumor infiltrating lymphocytes etc.

The disclosure also covers methods to isolate hematopoietic stem cellsfor cellular and gene therapy applications using method that include oneor more of (1) physical stress (e.g., hyperthermia), (2) nutritionaland/or metabolic stress including, but not limited to, serum starvationand/or growth factor starvation; (3) chemical (e.g., exposure tochemotoxic compounds); and (4) exposure to Pgp-transported phototoxiccompounds.

The disclosure also demonstrates that Pgp expression correlates with thedegree of stem-ness of a T or NK cell so that the cells with the highestlevel of Pgp expression are likely to be the most primitive (or moststem like).

“Treatment” and “treating,” as used herein refer to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition,prevent the pathologic condition, pursue or obtain beneficial results,or lower the chances of the individual developing the condition even ifthe treatment is ultimately unsuccessful. Those in need of treatmentinclude those already with the condition as well as those prone to havethe condition or those in whom the condition is to be prevented.

“Tumor,” as used herein refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

As mentioned Sukumar et al. taught that robust lymphocyte wereidentifiable based on low mitochondrial membrane potential. In contrast,the present disclosure provides methods and compositions to identifyrobust lymphocyte cells using markers and methods that are easier andclinically feasible. The disclosure demonstrates that P-glycoprotein(P-gp or Pgp) expression is a strong indicator of lymphocytedifferentiation and biological activity. Moreover, the disclosureprovides various dyes, chemotherapeutic agents, nutritional and/ormetabolic and physical stresses useful for selecting robust lymphocytecell types.

The cells and methods described herein use, in some cases, one or moremarkers wherein at least one marker is Pgp. A molecule is a “marker” ofa desired cell type if it is found on a sufficiently high percentage ofcells of the desired cell type, and found on a sufficiently lowpercentage of cells of an undesired cell type. One can achieve a desiredlevel of purification of the desired cell type from a population ofcells comprising both desired and undesired cell types by selecting forcells in the population of cells that have the marker. A marker can bedisplayed on, for example, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 99% or more of the desired cell type, and canbe displayed on fewer than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%,5%, 1% or fewer of an undesired cell type.

In one embodiment, the disclosure provides Pgp⁺ cells, individually orin populations. The term “isolated” or “purified” when referring to acell(s) of the disclosure means cells that are substantially free ofcells lacking the phenotypic marker (e.g., Pgp⁻) or vice versa. Inparticular embodiments, the cells are at least 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% free of acontaminating cell types (e.g., Pgp⁺ or Pgp⁻ cells as the case may be).In another embodiment, the isolated cells also are substantially free ofsoluble, naturally occurring molecules. A Pgp⁺ cell of the disclosure,for example, can be 99%-100% purified by, for example, flow cytometry(e.g., FACS analysis), as discussed herein.

In one embodiment, the disclosure provides an enriched population ofPgp⁺ or Pgp⁻ cells (depending upon the desired selection criteria). An“enriched population of cells” is one wherein a desired cell-type of thedisclosure has been partially separated from other cell types, such thatthe resulting population of cells has a greater concentration of Pgp⁺ orPgp⁻ cells than the original population of cells (they type of desiredcell with depend upon whether you are selecting “for” or “against” Pgpexpression). The enriched population of cells can have greater thanabout a 1.5-fold, 2-fold, 10-fold, 100-fold, 500-fold, 1,000-fold,2,000-fold, 3,000-fold, 4,000-fold, 5,000-fold, 6,000-fold, 7,000-fold,8,000-fold, 9,000-fold, 10,000-fold or greater concentration of, e.g.,Pgp⁺ cells than the original population had prior to separation. Pgp⁺cells of the disclosure can, for example, make up at least 5%, 10%, 15%,20%, 35%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 99% or more of the enriched population of stem cells. Theenriched population of cells may be obtained by, for example, selectingagainst cells displaying lacking a Pgp marker. Alternatively, or inaddition to, the enrichment for the expression of a marker, the loss ofexpression of a marker may also be used for enrichment. For example,lack of expression of a marker (e.g., Pgp) can be used to select cells.

In another embodiment, the disclosure uses chemotherapeutic agents toselect robust lymphocyte cells. As mentioned above, Pgp is a multidrugresistance protein that pumps a broad range of substrates, includingharmful substrates such as chemotherapeutics, out of cells. Thus, cellsthat express Pgp including high levels of Pgp are more resistant tochemotherapeutics. Accordingly, contacting a population of cellsexpressing various levels of Pgp with a chemotherapeutic(s) will selectfor those cells that have higher expression levels of Pgp compared tothose with no or low level expression of Pgp.

Similarly, dyes (e.g., photosensitive dyes) can be used in a similarfashion. In this embodiment, cells that express or over express Pgp willtend to pump out (or exclude) the dye compared to cells with low or noPgp expression. Dye-containing cells can be separate fromnon-dye-containing cells. Moreover, if the dye is toxic or can bephotoactivatated, cells that contain the dye can be killed thusretaining the Pgp expressing cells.

The disclosure also demonstrates that exposing cells to physicalstresses, such as hyperthermic conditions, can provide selection. Forexample, exposing lymphocytes to higher temperatures for short periodsof time provides a selective advantage to cells that express or overexpress Pgp compared to cells that do no expression Pgp.

The disclosure also demonstrates that exposing cells to serum-starvationand growth factor-starvation conditions can provide enrichment. Forexample, exposing peripheral blood stem cells to serum starvation forshort periods of time results in enrichment of CD34+ cells that expressor coexpress Pgp.

As disclosed herein, T cells expressing Pgp have better capacity forlong-term survival and ongoing effector function after adoptivetransfer. Thus, in one embodiment, expression and/or activity ofP-glycoprotein can be used to select lymphocytes for adoptive cellulartherapy including, but not limited to, genetic modification by CAR toproduce CAR-T cells, TCRs and chimeric TCRs. Since P-glycoprotein is acell surface protein and there a number of antibodies available againstit, the current disclosure provides a method of selecting lymphocytesfor adoptive cell therapy based on P-glycoprotein expression. TheP-glycoprotein positive cells of the disclosure can be isolated using anumber of techniques known in the art, such as magnetic activated cellsorting (MACS), in addition to conventional flow sorting. Thesetechniques have the advantage over the method proposed by Sukumar et al.in that they are quick, economical and amenable to clinical applicationas a number of systems for MACS are already in clinical use. Forexample, Miltenyi Biotech markets a system for MACS that has FDAapproval and is in clinical use in several centers.

P-glycoprotein expression can also be combined with positive andnegative selection of other markers (e.g., CD8, CD4, CD62L, CD44, etc.)to further enrich lymphocytes with stem-like property and/or to enrichfor stem-like T cells belonging to different subsets (e.g. cytotoxic,helper, Treg, etc.) for adoptive cellular therapy.

Provided herein are methods for isolating cells suitable for adoptivecell therapy. In one embodiment the methods comprise obtaining a sample,enriching the sample for T cells, mononuclear cells, NK cells, and/orstem cells and isolating p-glycoprotein positive (Pgp⁺) T cells,mononuclear cells, NK cells and/or stem cells from the enriched sample,so as to obtain a fraction enriched in Pgp⁺ T cells, NK cells, and/orstem cells suitable for adoptive cell transfer therapy. In someembodiments, the step of enriching the sample for T cells, mononuclearcells, NK cells and/or stem cells can be omitted. In some embodiments,isolating the Pgp⁺ T cells, NK cells and/or stem cells from the samplecomprises exposing the sample to at least one primary antibody orantibody-like moiety specific to p-glycoprotein. In some embodiments,the at least one primary antibody or antibody-like moiety is conjugatedto at least one fluorescent label or at least one magnetic label. Insome embodiments, the methods further comprise optionally staining thesample with at least one secondary antibody. In some embodiments, the atleast one secondary antibody is conjugated to at least one fluorescentlabel or at least one magnetic label. In some embodiments, isolating ofthe Pgp⁺ T cells, NK cells and/or stem cells from the sample isperformed by any one or more methods selected from immunofluorescentmethods, immunomagnetic methods, immunoaffinity methods or combinationsthereof. In some embodiments, isolating of the Pgp⁺ T cells, NK cellsand/or stem cells from the sample is performed by any one or moremethods selected from flow cytometry, magnetic activated cell sorting,biotin-streptavidin based affinity purification or combinations thereof.In some embodiments, Pgp⁺ T cells, NK cells and/or stem cells can beisolated from a sample (e.g. blood, bone marrow, leukopheresis sample,or peripheral blood mononuclear cells) in a single step by simultaneouslabeling with fluorochrome-conjugated antibodies against Pgp and othercellular markers associated with the cell type (e.g., T cell marker(s)such as CD3) followed by sorting for Pgp+/marker² (e.g., followed bysorting for Pgp⁺/CD3⁺ T cells) by flow sorting. In some embodiments,Pgp⁺ T cell subsets can be isolated from a sample (e.g. blood, bonemarrow, leukopheresis sample, or peripheral blood mononuclear cells) ina single step by simultaneous labeling with fluorochrome-conjugatedantibodies against Pgp and T cell subset marker(s) (e.g. CD4, CD8, etc.)followed by sorting for Pgp⁺/CD4⁺ or Pgp⁺/CD8⁺ T cells by flow sorting.

In addition to separation of Pgp expressing cells based on surfacelabeling with Pgp antibodies, the disclosure provides methods ofisolation/purification/enrichment of lymphocytes for adoptive cellulartherapy based on Pgp activity. A number of cytotoxic drugs, such asVincristine, vinblastin, doxorubicin, daunorubicin, taxol, paclitaxol,etoposide, mitoxantrone, actinomycin-D, etc. are substrates of Pgp.Therefore, Pgp-expressing cells can be enriched by exposing T cells toappropriate concentration of the above drugs that will kill Pgp-negativecells, thus isolating/purifying/enriching Pgp⁺ cells.

In another embodiment, the disclosure provides a method for isolatingcells suitable for adoptive cell therapy, comprising obtaining a sample,enriching the sample for T cells, contacting the sample with at leastone cytotoxic drug at a concentration appropriate to kill Pgp⁻ T cells,and isolating Pgp⁺ T cells from the enriched sample, so as to isolatecells suitable for adoptive cell transfer therapy. In anotherembodiment, the disclosure provides a method for isolating cellssuitable for adoptive cell therapy, comprising obtaining a sample,enriching the sample for mononuclear cells, contacting the sample withat least one cytotoxic drug at a concentration appropriate to killPgp-mononuclear cells, and isolating Pgp⁺ mononuclear cells from theenriched sample, so as to isolate cells suitable for adoptive celltransfer therapy. In some embodiments, the step of enriching the samplefor T cells or mononuclear cells can be omitted prior to contain thecells with a cytotoxic drug. In some embodiments, the cytotoxic drugsare any one or more of vincristine, vinblastin, doxorubicin, or taxol,or combinations thereof. In any of the foregoing embodiments, the cellpopulation enriched for Pgp⁺ cells can be further processed usingantibodies to markers on the cells (e.g., anti-Pgp antibodies), dyes, orhyperthermic conditions. In some embodiments, isolating the Pgp⁺ T cellsfrom the sample comprises exposing the sample to at least one primaryantibody or antibody like moiety specific to p-glycoprotein. In someembodiments, the at least one primary antibody or antibody like moietyis conjugated to at least one fluorescent label or at least one magneticlabel. In some embodiments, the methods further comprise optionallystaining the sample with at least one secondary antibody. In someembodiments, the at least one secondary antibody is conjugated to atleast one fluorescent label or at least one magnetic label. In someembodiments, isolating of the Pgp⁺ T cells from the sample is performedby any one or more methods selected from immunofluorescent methods,immunomagnetic methods, or combinations thereof. In some embodiments,isolating of the Pgp-positive T cells from the sample is performed byany one or more methods selected from flow cytometry, magnetic activatedcell sorting, or combinations thereof.

In addition, Pgp expressing cells can be purified/enriched/isolatedusing photodynamic dye treatment. For example, Pgp expression cells canbe purified/enriched/isolated with rhodamine analogs that aredifferentially retained between Pgp⁺ and Pgp⁻ cells and becomephototoxic on exposure to visible light. This method can be used aloneor in combination with other methods of selection described herein.Exemplary rhodamine and rhodamine derivatives are known and include, forexample, derivatives selected from the group consisting of4,5-dibromorhodamine 123(2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid methylester hydrochloride); 4,5-dibromorhodamine 123(2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid ethylester hydrochloride); 4,5-dibromorhodamine 123(2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid octylester hydrochloride); 4,5-dibromorhodamine 110 n-butyl ester(2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid n-butylester hydrochloride); Rhodamine B n-butyl ester (2-(6-ethylamino-3-ethyl imino-3H-xanthen-9-yl)-benzoic acid n-butyl esterhydrochloride); and photoactivable derivatives thereof; wherebyphotoactivation of said derivatives induces cell killing whileunactivated derivatives are substantially non-toxic to cells. Otherphototoxic compounds that are substrate of Pgp have been described inthe literature as well and can be used in the method of the currentdisclosure. For example, using a rhodamine or rhodamine analog, apopulation of cells can be contacted with a rhodamine or rhodamineanalog under sufficient concentrations and time that allow for cells totake up the rhodamine or rhodamine analog. As described herein andabove, Pgp expressing cells will pump the rhodamine or rhodamine analogout of the cell, while Pgp⁻ or cells having reduced Pgp expression willretain the rhodamine or rhodamine analog. The population is thencontacted with a light of a wavelength that causes ablation of cellscontaining the rhodamine or rhodamine analog (i.e., cells lacking orhaving reduced Pgp expression). Thus, the population will be enrichedfor Pgp⁺ cells by photo-ablation of Pgp⁻ cells.

Due to the specific retention of the rhodamine 123 (Rh123) class of dyesby Pgp⁻ cells and the concomitant lack of their accumulation by thelymphocytes with stem-like phenotype (e.g., by Pgp⁺), the disclosureprovides a method for the use of these dyes for in vivo or in vitrophotodynamic therapy to enrich lymphocytes for adoptive cell therapy.

Since low staining with TMRM, Rh123 and DiOC2(3) correlates with and/oris primarily due to Pgp mediated efflux and is not solely due to lowmitochondrial membrane potential, the disclosure teaches an optimizedprotocol for isolation of Pgp expressing cells by optimizing Pgpmediated efflux. For example, by performing the assay at 37° C. and byallowing more time for Pgp mediated efflux of the dyes, a betterdifferentiation can be obtained between Pgp⁺ and Pgp⁻ cells for thepurpose of adoptive cellular therapy.

In another embodiment, the disclosure provides a method for isolatingcells suitable for adoptive cell therapy, comprising obtaining a sample,enriching the sample for T cells, NK cells, stem cells, and/ormononuclear cells, contacting the sample with at least one phototoxiccompound, exposing the sample to a visible light source sufficient toactivate the at least one phototoxic compound so as to kill Pgp⁻ Tcells, NK cells, stem cells, and/or mononuclear cells, and isolatingPgp⁺ T cells, NK cells, stem cells, and/or mononuclear cells from theenriched sample, so as to isolate cells suitable for adoptive celltransfer therapy. This method can be used alone or in combination withother methods for Pgp⁺ cell enrichment. In some embodiments, the step ofenriching the sample for T cells, NK cells, stem cells, and/ormononuclear cells can be omitted. In some embodiments, the phototoxiccompounds are any one or more of2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid methylester hydrochloride,2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid ethyl esterhydrochloride, 2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoicacid octyl ester hydrochloride,2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid n-butylester hydrochloride, 2-(6-ethyl amino-3-ethylimino-3H-xanthen-9-yl)-benzoic acid n-butyl ester hydrochloride, orderivatives thereof or combinations thereof. For example, using aphototoxic, a population of cells can be contacted with a phototoxiccompound under sufficient concentrations and time that allow for cellsto take up the phototoxic compound. As described herein and above, Pgpexpressing cells will pump the phototoxic compound out of the cell,while Pgp⁻ or cells having reduced Pgp expression will retain thephototoxic compound. The population is then contacted with a light of awavelength that causes ablation of cells containing the phototoxiccompound (i.e., cells lacking or having reduced Pgp expression). Thus,the population will be enriched for Pgp⁺ cells by photo-ablation of Pgp⁻cells.

In another embodiment, physical methods, such as temperature exposure,can be used to select for CD34⁺ and/or Pgp⁺ cells. In this embodiment,the disclosure provides a method for isolating cells suitable foradoptive cell therapy, comprising obtaining a sample, enriching thesample for T cells, NK cells, stem cells, and/or mononuclear cells,culturing the cells and exposing the population of cells to hyperthermiaso as to kill Pgp⁻ T cells, NK cells, stem cells, and/or mononuclearcells, and isolating/enriching for Pgp⁺ T cells, NK cells, stem cells,and/or mononuclear cells. In one embodiment, the cells are exposed to atemperature from about 40-42° C. for 2-4 hours. In one embodiment, thetemperature is 42-43° C. In another embodiment, the temperature is 42°C. for 2-3 hours. In another embodiment, the temperature is 43° C. for2-3 hours. This method can be used alone or in combination with othermethods for Pgp⁺ cell enrichment. In some embodiments, the step ofenriching the sample for T cells, NK cells, stem cells, and/ormononuclear cells can be omitted. For example, using a hyperthermictemperature during culturing, a population of cells can be enriched forPgp⁺ cells.

In another embodiment, temperature exposure can be used to select forPgp⁺/CD34⁺ cells. In this embodiment, the disclosure provides a methodfor isolating cells suitable for adoptive cell therapy, comprisingobtaining a sample, enriching the sample for T cells or mononuclearcells, culturing the cells and exposing the population of cells tohyperthermia so as to kill Pgp⁻ T, NK or mononuclear cells, andisolating/enriching for Pgp⁺ stem cells and mononuclear cells. In oneembodiment, the cells are exposed to a temperature from about 40-42° C.for 2-4 hours. In one embodiment, the temperature is 42-43° C. Inanother embodiment, the temperature is 42° C. for 2-3 hours. In anotherembodiment, the temperature is 43° C. for 2-3 hours. The cells can thenbe “panned” for CD34⁺ marker. This method can be used alone or incombination with other methods for Pgp⁺/CD34⁺ cell enrichment. Forexample, using a hyperthermic temperature during culturing, a populationof cells can be enriched for Pgp⁺/CD34⁺ cells.

In some embodiments, where cytotoxic agents are used (e.g.,chemotherapeutics and/or photoactive dyes and/or other agents) and/orphysical stress (such as hyperthermic treatment) and/ornutritional/metabolic stress (e.g., serum- or growth factorstarvation/depletion) are used for isolating the Pgp⁺ cells from thesample the method can further include, after or prior to the abovemethods, exposing the sample to at least one primary antibody orantibody like moiety specific to p-glycoprotein. In some embodiments,the at least one primary antibody or antibody like moiety is conjugatedto at least one fluorescent label or at least one magnetic label orbiotin. In some embodiments, the methods further comprise optionallystaining the sample with at least one secondary antibody. In someembodiments, the at least one secondary antibody is conjugated to atleast one fluorescent label or at least one magnetic label or biotin. Insome embodiments, isolating of the Pgp⁺ T cells from the sample isperformed by any one or more methods selected from immunofluorescentmethods, immunomagnetic methods, immunoaffinity methods or combinationsthereof. In some embodiments, isolating of the Pgp⁺ T cells from thesample is performed by any one or more methods selected from flowcytometry, magnetic activated cell sorting, biotin-streptavidin basedcell sorting or combinations thereof. Although not necessary, but usinga combination of purification methods the enrichment of Pgp⁺ cells canbe further improved and/or optimized. In some embodiments, the fractionenriched in Pgp⁺ T cells contains less than 50% Pgp⁻ T cells. In someembodiments, the fraction enriched in Pgp⁺ T cells contains less than40% Pgp⁻ T cells. In some embodiments, the fraction enriched in Pgp⁺ Tcells contains less than 30% Pgp⁻ T cells. In some embodiments, thefraction enriched in Pgp⁺ T cells contains less than 20% Pgp⁻ T cells.In some embodiments, the fraction enriched in Pgp⁺ T cells contains lessthan 10% Pgp⁻ T cells. In some embodiments, the fraction enriched inPgp⁺ T cells contains less than 5% Pgp⁻ T cells. In some embodiments,the fraction enriched in Pgp⁺ T cells contains less than 1% Pgp⁻ Tcells.

In some embodiments, the fraction enriched in Pgp⁺ mononuclear cellscontains less than 50% Pgp⁻ mononuclear cells. In some embodiments, thefraction enriched in Pgp⁺ mononuclear cells contains less than 40% Pgp⁻mononuclear cells. In some embodiments, the fraction enriched in Pgp⁺mononuclear cells contains less than 30% Pgp⁻ mononuclear cells. In someembodiments, the fraction enriched in Pgp⁺ mononuclear cells containsless than 20% Pgp⁻ mononuclear cells. In some embodiments, the fractionenriched in Pgp⁺ mononuclear cells contains less than 10% Pgp⁻mononuclear cells. In some embodiments, the fraction enriched in Pgp⁺mononuclear cells contains less than 5% Pgp⁻ mononuclear cells. In someembodiments, the fraction enriched in Pgp⁺ mononuclear cells containsless than 1% Pgp⁻ mononuclear cells.

In addition to Pgp, a number of other drug transporters (includingbreast cancer resistance protein) are selectively expressed on thesurface of stem cells. Antibodies and substrates of these proteins canbe also used to enrich lymphocytes with stem like phenotype for thepurpose of adoptive cellular therapies. Thus, using selection techniquesfor these markers in combination with the selection techniques describedherein for Pgp⁺ cell can improve cell-selection processing techniques.

Once the Pgp expressing cells have been enriched by any of the abovemethods, the cells can be used for gene modification with CAR, TCR,chimeric TCR, synthetic immune receptor, TRuC™ T cell platform, Artemis™T cell platform or other methods for the purpose of adoptive cellulartherapy. They could be also used without gene modification, for examplefor immunization with T cell antigens. In some embodiments, the Pgp⁺ Tcells obtained by the methods described herein are genetically modifiedfor use in adoptive cell therapy. In exemplary embodiments, the Pgp⁺ Tcells are genetically modified to express at least one chimeric antigenreceptor, T cell receptor, chimeric T cell receptor, synthetic immunereceptor, TRuC™ T cell platform, Artemis™ T cell platform fortherapeutic uses, such as for treating cancer.

In some embodiments, the genetically modified Pgp⁺ T cells according toany of the methods disclosed herein may be used in treating cancer,infection or immune disorders. Accordingly, in various embodiments, thedisclosure provides methods for treating cancer or immune disorders in asubject, comprising providing genetically modified Pgp⁺ cells describedherein, and administering a therapeutically effective amount of thecells to the subject so as to treat cancer.

In some embodiments, the cancer is B-cell lymphomas, T cell lymphoma,skin cancer, testicular cancer, endocrine cancer, cancer of unknownprimary site, rectal cancer, anal cancer, esophageal cancer, brain tumor(e.g., glioblastoma multiforme), breast cancer, colon cancer, lungcancer, hepatocellular cancer, gastric cancer, pancreatic cancer,cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer ofthe urinary tract, cancer of reproductive tract, thyroid cancer, renalcancer, carcinoma, melanoma, head and neck cancer, brain cancer,prostate cancer, or leukemia. In some embodiments, the cancer is B-celllymphomas, T cell lymphomas, myeloma, myelodysplastic syndrome, skincancer, brain tumor, breast cancer, colon cancer, rectal cancer,esophageal cancer, anal cancer, cancer of unknown primary site,endocrine cancer, testicular cancer, lung cancer, hepatocellular cancer,gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer,liver cancer, bladder cancer, cancer of the urinary tract, cancer ofreproductive organs, thyroid cancer, renal cancer, carcinoma, melanoma,head and neck cancer, brain cancer, prostate cancer, or leukemia.

In some embodiments, the genetically modified Pgp⁺ cells describedherein are administered simultaneously or sequentially with achemotherapeutic agent. In some embodiments, the chemotherapeutic agentis selected from alkylating agents, alkyl sulfonates, aziridines,ethylenimines, methylamelamines, acetogenins, a camptothecin,bryostatin, callystatin, CC-1065, cryptophycins, dolastatin,duocarmycin, eleutherobin, pancratistatin, a sarcodictyin, spongistatin,nitrogen mustards, nitrosureas, antibiotics, dynemicin, bisphosphonates,an esperamicin, neocarzinostatin chromophore and related chromoproteinenediyne antibiotic chromophores, aclacinomysins, actinomycin,authramycin, azaserine, bleomycins, cactinomycin, carabicin,caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin,detorubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, deoxydoxorubicin,epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins,anti-metabolites, folic acid analogues, purine analogs, pyrimidineanalogs, androgens, anti-adrenals, folic acid replenisher, aceglatone,aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine,bestrabucil, bisantrene, edatraxate, defofamine, demecolcine,diaziquone, elformithine, elliptinium acetate, an epothilone, etoglucid,gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids,mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin,phenamet, pirarubicin, losoxantrone, podophyllinic acid,2-ethylhydrazide, procarbazine, razoxane, rhizoxin, sizofuran,spirogermanium, tenuazonic acid, triaziquone,2,2′,2″-trichlorotriethylamine, trichothecenes, urethane, vindesine,dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman,gacytosine, arabinoside, cyclophosphamide, thiotepa, taxoids,chloranbucil, 6-thioguanine, mercaptopurine, methotrexate, platinumanalogs, vinblastine, platinum, etoposide (VP-16), ifosfamide,mitoxantrone, vincristine, vinorelbine, novantrone, teniposide,edatrexate, daunomycin, aminopterin, xeloda, ibandronate, irinotecan,topoisomerase inhibitor RFS 2000; difluoromethylornithine, retinoids,capecitabine, combretastatin, leucovorin, oxaliplatin, lapatinib,inhibitors of PKC-alpha, Raf, H-Ras, EGFR and VEGF-A that reduce cellproliferation, and pharmaceutically acceptable salts, acids orderivatives of any of the above, or combinations thereof.

In addition to enriching Pgp-expressing immune cells for the purpose ofadoptive T cell therapies, the disclosure also teaches methods todeplete Pgp-expressing immune cells from stem cell grafts given topatients undergoing allogeneic stem cell transplant to lower the risk ofGraft versus Host disease. The disclosure also teaches methods todeplete Pgp-expressing immune cells from donor T cell given toimmunodeficient patients to boost their immunity against infectionswhile lowering the risk of Graft-vs-Host Disease (GVHD). One of skill inthe art would recognize that methods described herein for killing orablation of Pgp⁻ cells (i.e., the selective killing of Pgp⁻) would notbe useful for depleting a sample of Pgp⁺ cells. Rather methods that arecapable of selectively killing or removing Pgp⁺ cells while leaving thePgp-cells intact would be used.

Graft-versus-host disease (GVHD) is the main cause of mortality and amajor limitation to the early and widespread use of allogeneic stem celltransplantation (SCT), a treatment that often represents the onlycurative option for numerous patients with malignant diseases andhereditary metabolic disorders. Depletion of T cells capable ofrecognizing and mounting an immune response toward host cells from stemcell grafts can reduce or even eliminate GVHD. However, the eliminationof T cells also results in delayed T-cell reconstitution and, thus, anincreased rate of infection, particularly with viral agents such ascytomegalovirus, herpes zoster, and Epstein-Barr virus. In addition, theeradication of mature T cells is associated with an increased risk ofgraft rejection and an increased incidence of relapse of malignantdisease. Thus, T cells are required early after allogeneictransplantation and depleting the graft of its T-cell content is not anideal approach to prevention of complications after transplantation.Although new immunosuppressive agents offer options to decrease theincidence and severity of GVHD, most of the time these strategies areonly partially effective and may also increase the incidence of viraland fungal infections and other adverse effects of profoundimmunosuppression.

To provide a solution to this problem, selective inactivation orelimination of alloreactive donor T lymphocytes could allow early immunerecovery and response toward infectious agents, and potentially preservegraft-versus-leukemia (GVL) activity. In addition, a strategy toselectively eliminate immunoreactive T cells could represent animportant advance for the treatment of a large number of patients withautoimmune disorders. In this procedure, stimulation of T cells withmitogens or allogeneic major histocompatibility complex-mismatched cellsresulted in the preferential retention of the TH9402rhodamine-derivative in activated T cells, both CD4 and CD8.Photodynamic cell therapy of TH9402-exposed T cells led to the selectiveelimination of immunoreactive T-cell populations.

Based on the discovery that Pgp is expressed on T stem cells and itsexpression is lost upon T cell activation, Pgp⁻ cells in a donor havebeen already exposed to an antigen. The likely antigens for such Pgp⁻cells in a healthy donor are likely to be common pathogens, such asviruses and fungi. Thus, Pgp⁻ cells may confer immunity to pathogenscommonly encountered in the environment. In contrast, Pgp⁺ cells arelikely to contain naïve cells that can cause GVHD when given to a donor.Therefore, elimination of Pgp⁺ cells from a graft may enrich for T cellsthat can confer immunity while sparing GVHD. In the present disclosurethe method depletes Pgp-expressing T cells obtained from a donor. Thus,the present method involves depletion of Pgp⁺ cells. In contrast toother methods, the method of present disclosure does not involvestimulation of donor T cells with mitogen or MHC mismatched host cells.Finally, the method of the present disclosure involves depletion of Pgpexpressing cells by staining with Pgp antibody or an antibody likemoiety (e.g. scFv, vHH, affibody, nanobody, Fab fragment, Darpins etc.).In the method described herein, the depletion of Pgp expressing cells isachieved by staining T cells with more than 1 antibody or antibody likemoieties directed against different epitopes on the extracellular domainof Pgp. Since Pgp is also expressed on normal hematopoietic stem cells,depletion of Pgp expressing cells would potentially deplete stem cellsfrom the graft. As such, in one method of the disclosure, stem cells arefirst positively selected from the graft using an antibody against CD34antigen. This can be achieved using a commercially available CD34isolation system (Miltenyi). Subsequently, the CD34-negative flowthrough fraction of the graft is depleted of Pgp expressing cells byimmunostaining with Pgp antibody or a cocktail of antibodies. The CD34⁺stem cell fraction is then administered with CD34-/Pgp⁻ T cell fractionto the patient undergoing allogeneic bone marrow, peripheral blood stemcell, or umbilical cord stem cell transplant. The Pgp⁻ T cell fractioncan also be given to the patient at a later time than CD34⁺ stem cellinfusion.

Another application of the disclosure is for adoptive cellular therapyin immunodeficient patients, such as allogeneic stem cell transplant(including umbilical cord transplant) recipients, who areimmunodeficient and have become infected due to poor T cellreconstitution. Administration of a T cell population that is enrichedfor T cells capable of conferring immunity to viral, bacterial andfungal pathogens, but have limited capacity for alloreactivity or tocause GVHD, is highly desirable in this setting. Therefore,administration of Pgp⁻ T cell population obtained from the donor(primary donor or even third party donors) to such patients will protectagainst infectious agents while not significantly increasing the risk ofGVHD.

In one embodiment, the disclosure provides a method for isolating Pgp⁻ Tcells suitable for adoptive cell transfer therapy, comprising obtaininga sample, enriching the sample for T cells, and depleting Pgp⁺ T cellsfrom the sample, so as to obtain a fraction enriched in Pgp⁻ T cellssuitable for adoptive cell transfer therapy. In some embodiments,depleting the Pgp⁺ T cells from the sample comprises exposing the sampleto at least one primary antibody or antibody like moiety specific top-glycoprotein. In some embodiments, the at least one primary antibodyor antibody like moiety is conjugated to at least one fluorescent labelor at least one magnetic label or biotin. In some embodiments, themethods further comprise optionally staining the sample with at leastone secondary antibody. In some embodiments, the at least one secondaryantibody is conjugated to at least one fluorescent label or at least onemagnetic label or biotin. In some embodiments, depleting of the Pgp⁺ Tcells from the sample is performed by any one or more methods selectedfrom immunofluorescent methods, immunomagnetic methods, immunoaffinitymethods or combinations thereof. In some embodiments, depleting of thePgp⁺ T cells from the sample is performed by flow cytometry, magneticactivated cell sorting, Biotin-streptavidin based immunoaffinity cellsorting or combinations thereof. In some embodiments, the fractionenriched in Pgp⁻ T cells contains less than 50% Pgp⁺ T cells. In someembodiments, the fraction enriched in Pgp-negative T cells contains lessthan 40% Pgp⁺ T cells. In some embodiments, the fraction enriched inPgp⁻ T cells contains less than 30% Pgp⁺ T cells. In some embodiments,the fraction enriched in Pgp⁻ T cells contains less than 20% Pgp⁺ Tcells. In some embodiments, the fraction enriched in Pgp⁻ T cellscontains less than 10% Pgp⁺ T cells. In some embodiments, the fractionenriched in Pgp⁻ T cells contains less than 5% Pgp⁺ T cells. In someembodiments, the fraction enriched in Pgp⁻ T cells contains less than 1%Pgp⁺ T cells.

In some embodiments, the disclosure provides a method for the treatmentof infection in an immunodeficient HIV/AIDS subject, comprisingproviding a composition comprising a population of Pgp⁻ T cells isolatedby the methods described herein and administering a therapeuticallyeffective amount of the composition to the subject so as to treat theinfection. The infection may be viral infection (e.g. cytomegalovirus,adenovirus or BK virus), a bacterial infection (e.g. mycobacterium,enterococcus), fungal infection (e.g. mucor or aspergillus) or protozoaninfection (e.g. toxoplasmosis).

In some embodiments, the disclosure provides a method for the treatmentof infection in a subject undergoing an allogeneic stem cell transplant,comprising providing a composition comprising a population of Pgp⁻ Tcells isolated by the methods described herein and administering atherapeutically effective amount of the composition comprising the Pgp⁻cells to the subject so as to treat the infection. The infection may beviral infection (e.g. cytomegalovirus, adenovirus or BK virus), abacterial infection (e.g. mycobacterium, enterococcus), fungal infection(e.g. mucor or aspergillus) or protozoan infection (e.g. toxoplasmosis).

In some embodiments, the disclosure provides a method for reducinggraft-versus-host disease in a subject undergoing an allogeneic stemcell transplant, comprising providing a composition comprising apopulation of Pgp⁻ T cells isolated by the methods described herein andadministering a therapeutically effective amount of the compositioncomprising the Pgp⁻ T cells to the subject so as to reducegraft-versus-host disease. In some embodiments, the transplant is anallogeneic bone marrow transplant, an allogeneic peripheral blood stemcell transplant, a haploidentical bone marrow transplant, ahaploidentical peripheral blood stem cell transplant, or an umbilicalcord stem cell transplant.

In various embodiments, the disclosure provides pharmaceuticalcompositions comprising a population of the Pgp⁺ T cells isolated by anyof the methods disclosed herein, and at least one pharmaceuticallyacceptable carrier. In some embodiments, the Pgp⁺ T cells aregenetically modified as described herein. In some embodiments, thepharmaceutical compositions comprising the genetically modified Pgp⁺ Tcells are used in cancer therapies as described herein. In someembodiments, the pharmaceutical compositions comprising the geneticallymodified Pgp⁺ T cells are used in the treatment of infectious and immunedisorders as described herein.

In various embodiments, the disclosure provides pharmaceuticalcompositions comprising a population of Pgp⁻ T cells isolated by any ofthe methods described herein, and at least one pharmaceuticallyacceptable carrier. In various embodiments, the pharmaceuticalcompositions comprising the Pgp⁻ T cells isolated by the methodsdescribed herein are used in therapies including but not limited toreduction of graft-versus-host disease in a subject undergoingallogeneic transplant (including stem cell transplant), treatment of aninfection in a subject in need thereof and/or treatment of an infectionin a HIV/AIDS subject, wherein the infection may be viral infection(e.g. cytomegalovirus, adenovirus or BK virus), a bacterial infection(e.g. mycobacterium, enterococcus), fungal infection (e.g. mucor oraspergillus) or protozoan infection (e.g. toxoplasmosis).

The pharmaceutical compositions according to the invention can containany pharmaceutically acceptable excipient. “Pharmaceutically acceptableexcipient” means an excipient that is useful in preparing apharmaceutical composition that is generally safe, non-toxic, anddesirable, and includes excipients that are acceptable for veterinaryuse as well as for human pharmaceutical use. Such excipients may besolid, liquid, semisolid, or, in the case of an aerosol composition,gaseous. Examples of excipients include but are not limited to starches,sugars, microcrystalline cellulose, diluents, granulating agents,lubricants, binders, disintegrating agents, wetting agents, emulsifiers,coloring agents, release agents, coating agents, sweetening agents,flavoring agents, perfuming agents, preservatives, antioxidants,plasticizers, gelling agents, thickeners, hardeners, setting agents,suspending agents, surfactants, humectants, carriers, stabilizers, andcombinations thereof.

In various embodiments, the pharmaceutical compositions according to thedisclosure may be formulated for delivery via any route ofadministration. “Route of administration” may refer to anyadministration pathway known in the art, including but not limited toaerosol, nasal, oral, transmucosal, transdermal, parenteral or enteral.“Parenteral” refers to a route of administration that is generallyassociated with injection, including intraorbital, infusion,intraarterial, intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal. Via the parenteral route, thecompositions may be in the form of solutions or suspensions for infusionor for injection. Via the parenteral route, the compositions may be inthe form of solutions or suspensions for infusion or for injection. Viathe enteral route, the pharmaceutical compositions can be in the form ofgel capsules, syrups, suspensions, solutions, emulsions, microspheres orlipid vesicles or polymer vesicles. Typically, the compositions areadministered by injection. Methods for these administrations are knownto one skilled in the art. In another embodiment, the compositions canbe part of a tissue delivery device or implant. In such embodiments, thecells are allowed to grow and/or exist in a biocompatible implantablestructure (e.g., with in collagen matrix and the like). In anotherembodiment, the cells may be applied to a structure prior toimplantation (e.g., a stent, balloon, valve, pump etc.).

The pharmaceutical compositions according to the disclosure can containany pharmaceutically acceptable carrier. “Pharmaceutically acceptablecarrier” as used herein refers to a pharmaceutically acceptablematerial, composition, or vehicle that is involved in carrying ortransporting a compound of interest from one tissue, organ, or portionof the body to another tissue, organ, or portion of the body. Forexample, the carrier may be a liquid or solid filler, diluent,excipient, solvent, or encapsulating material, or a combination thereof.Each component of the carrier must be “pharmaceutically acceptable” inthat it must be compatible with the other ingredients of theformulation. It must also be suitable for use in contact with anytissues or organs with which it may come in contact, meaning that itmust not carry a risk of toxicity, irritation, allergic response,immunogenicity, or any other complication that excessively outweighs itstherapeutic benefits.

The pharmaceutical compositions according to the disclosure can also beencapsulated or prepared in an emulsion or syrup for oraladministration. Pharmaceutically acceptable solid or liquid carriers maybe added to enhance or stabilize the composition, or to facilitatepreparation of the composition. Liquid carriers include syrup, peanutoil, olive oil, glycerin, saline, alcohols and water. Solid carriersinclude starch, lactose, calcium sulfate, dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. The carrier may also include a sustained release material suchas glyceryl monostearate or glyceryl distearate, alone or with a wax.

The pharmaceutical compositions according to the invention may bedelivered in a therapeutically effective amount. The precisetherapeutically effective amount is that amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given subject. This amount will vary depending upon a variety offactors, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, for instance, by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly. Foradditional guidance, see Remington: The Science and Practice of Pharmacy(Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000). In oneembodiment, the pharmaceutical composition comprises Pgp⁺ or Pgp⁻ cellsat 1×10⁵ to 1×10⁸ cells/ml (or any value there between which isexpressly contemplated herein). The cells may be introduced directlyinto the peripheral blood or deposited within other locations throughoutthe body, e.g., a desired tissue, or on microcarrier beads in theperitoneum. For example, 10² to 10¹¹ cells can be transplanted in asingle procedure, and additional transplants can be performed asrequired.

Cells can be engineered using any of a variety of vectors including, butnot limited to, integrating viral vectors, e.g., retroviral vectors orlentiviral vectors or adeno-associated viral vectors; or non-integratingreplicating vectors, e.g., papilloma virus vectors, SV40 vectors,adenoviral vectors; or replication-defective viral vectors. Wheretransient expression is desired, non-integrating vectors and replicationdefective vectors may be used, since either inducible or constitutivepromoters can be used in these systems to control expression of the geneof interest. Where the vector is a non-integrating vector, such vectorscan be lost from cells by dilution, as desired. An example of anon-integrating vector includes Epstein-Barr virus (EBV) vector.Alternatively, integrating vectors can be used to obtain transientexpression, provided the gene of interest is controlled by an induciblepromoter. Other methods of introducing DNA into cells include the use ofliposomes, lipofection, electroporation, a particle gun, or by directDNA injection. Alternatively, cells can be engineered using transfectionof in vitro transcribed mRNA.

Conventional recombinant DNA techniques can be used in the methods ofthe disclosure. For example, conventional recombinant DNA techniques areused to introduce a desired polynucleotide into cells (e.g.,polynucleotides encoding a CAR). The precise method used to introduce apolynucleotide is not critical to the disclosure. For example, physicalmethods for the introduction of polynucleotides into cells includemicroinjection and electroporation or viral gene therapy. Chemicalmethods such as coprecipitation with calcium phosphate and incorporationof polynucleotides into liposomes are also standard methods ofintroducing polynucleotides into mammalian cells. For example, DNA orRNA can be introduced using standard vectors, such as those derived frommurine and avian retroviruses (see, e.g., Gluzman et al., 1988, ViralVectors, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) orlentiviral vector systems. Standard recombinant molecular biologymethods are well known in the art (see, e.g., Ausubel et al., 1989,Current Protocols in Molecular Biology, John Wiley & Sons, New York),and viral vectors for gene therapy have been developed and successfullyused clinically (Rosenberg, et al., 1990, N. Engl. J. Med, 323:370).Other methods, such as naked polynucleotide uptake from a matrix coatedwith DNA are also encompassed by the disclosure (see, for example, U.S.Pat. No. 5,962,427, which is incorporated herein by reference).

Any promoter may be used to drive the expression of the inserted gene.For example, viral promoters include but are not limited to the CMVpromoter/enhancer, SV40, papillomavirus, Epstein-Barr virus, elastingene promoter and beta-globin. If transient expression is desired,constitutive promoters are used in a non-integrating and/orreplication-defective vector. Alternatively, inducible promoters couldbe used to drive the expression of the inserted gene when necessary.Inducible promoters can be built into integrating and/or replicatingvectors. For example, inducible promoters include, but are not limitedto, metallothionien and heat shock protein.

The lymphocytes or progenitor lymphocytes of the disclosure can beisolated from a sample obtained from a mammalian subject. The subjectcan be any mammal (e.g., bovine, ovine, porcine, canine, feline, equine,primate), including a human. The sample of cells may be obtained fromany of a number of different sources including, for example, bonemarrow, fetal tissue (e.g., fetal liver tissue), peripheral blood,umbilical cord blood, healthy or diseased tissue (e.g., tumorinfiltrating lymphocytes) and the like.

Although the disclosure has exemplified in various embodiments Pgp⁺ andPgp⁻ T cells, the methods and compositions of the disclosure areapplicable to NK cells, Pgp⁺ hematopietic stem cells (e.g., CD34+/Pgp⁺hematopoietic stem cells) and the like. Thus, in any of the variousembodiments describe herein and throughout the specification the term “Tcell” can be replaced with “NK cell” or “CD34⁺ cell” etc. One of skillin the art will recognize that CD34⁺ cells are stem cells and thus haveadditional methods and compositions applications that extend beyond Tcell. For example, CD34⁺ cells can be isolated/purified using metabolicstarvation, hyperthermia and/or chemotoxic compounds and used forallogeneic or autologous stem cell transplantation with or withoutgenetic modifications to the cells.

In another embodiment, the disclosure provides methods of establishingand/or maintaining populations of cells, or the progeny thereof, as wellas mixed populations comprising various cells types as well assubpopulations (e.g., Pgp⁺ and/or Pgp⁻). Once a culture of cells or amixed culture of cells and/or progenitor cell (stem-like cells) isestablished, the population of cells is maintained and/or mitoticallyexpanded in vitro by passage to fresh medium as cell density dictatesunder conditions conducive to cell proliferation and maintenance.

Once cells or desired sub-population of cells of the disclosure havebeen established in culture, as described above, they may be maintainedor stored in cell “banks” comprising either continuous in vitro culturesof cells requiring regular transfer or cells which have beencryopreserved.

Cryopreservation of cells of the disclosure may be carried out accordingto known methods, such as those described in Doyle et al., (eds.), 1995,Cell & Tissue Culture: Laboratory Procedures, John Wiley & Sons,Chichester. For example, but not by way of limitation, cells may besuspended in a “freeze medium” such as, for example, culture mediumfurther comprising 15-20% fetal bovine serum (FBS) and 10%dimethylsulfoxide (DMSO), with or without 5-10% glycerol, at a density,for example, of about 4-10×10⁶ cells/ml. The cells are dispensed intoglass or plastic vials which are then sealed and transferred to afreezing chamber of a programmable or passive freezer. The optimal rateof freezing may be determined empirically. For example, a freezingprogram that gives a change in temperature of −1° C./min through theheat of fusion may be used. Once vials containing the cells have reached−80° C., they are transferred to a liquid nitrogen storage area.Cryopreserved cells can be stored for a period of years, though theyshould be checked at least every 5 years for maintenance of viability.

The cryopreserved cells of the disclosure constitute a bank of cells,portions of which can be withdrawn by thawing and then used to produce acell culture as needed. Thawing should generally be carried out rapidly,for example, by transferring a vial from liquid nitrogen to a 37° C.water bath. The thawed contents of the vial should be immediatelytransferred under sterile conditions to a culture vessel containing anappropriate medium. It is advisable that the cells in the culture mediumbe adjusted to an initial density of about 1-3×10⁵ cells/ml. Once inculture, the cells may be examined daily, for example, with an invertedmicroscope to detect cell proliferation, and subcultured, ifappropriate, as soon as they reach an appropriate density.

In another embodiment, the disclosure provides cell lines of Pgp⁺ cells.As used herein a “cell line” means a culture of cells of the disclosure,or progeny cells thereof, that can be reproduced for an extended periodof time, preferably indefinitely, and which term includes, for example,cells that are cultured, cryopreserved and re-cultured followingcryopreservation. As used herein a “culture” means a population of cellsgrown in a medium and optionally passaged accordingly.

The following examples are intended to illustrate particular embodimentsand not to limit the scope of the disclosure.

EXAMPLES Example 1

Tetramethylrhodamine methyl ester (TMRM) was purchased from Thermofisherand dissolved in DMSO. DiOC2(3) was purchased from Life Technologies anddissolved in methanol to produce 1 mg/ml stock solution. TH9402 wassynthesized by the Weill Cornell Medicine Milstein Chemistry CoreFacility and dissolved in DMSO to generate 100 mM stock. Unconjugatedand FITC-conjugated monoclonal antibody (MoAb) UIC2 (IgG2a) against anextracytoplasmic domain of human Pgp was obtained from Santa CruzBiotechnology. Fluorescein isothiocyanate- (FITC)-conjugated goatantimouse IgG was from Southern Biotechnology. Buffy coat cells wereobtained from healthy de-identified adult donors from the Blood Bank atChildren Hospital of Los Angeles and used to isolate peripheral bloodmononuclear cells (PBMC) by Ficoll-Hypaque gradient centrifugation. PBMCwere either used as such or used to isolate T cells using CD3 magneticmicrobeads (Miltenyi Biotech) and following the manufacturer'sinstructions. PBMC or isolated T cells were re-suspended in XVIVO medium(Lonza) supplanted with 10 ng/ml soluble anti-CD3, 10 ng/ml solubleanti-CD28 and 100 IU recombinant human-IL2 unless indicated otherwise.Cells were cultured at 37° C., in a 5% CO₂ humidified incubator, unlessindicated otherwise.

Approximately 15 million PBMC cells were stained with TMRM (20 nM finalconcentration) in XVIVO medium for 30 min at 37° C. in the absence orpresence of Reserpine (50 μl of 1 mM stock added to 5 ml of medium withcells to give final concentration of 10 μM) or cyclosporine (10 μl of 1mM stock added to 5 ml of medium with cells; final concentration=2 μM).All subsequent steps were done with cells maintained at 4° C. in dark.Cells were centrifuged and cell pellets were blocked with 200 μl ofhuman AB serum for 1 hr at 4° C. Cells were washed with ice cold PBScontaining 1% FCS, and divided into 3 tubes. Cells in each tube werecentrifuged and cell pellets incubated for approximately 2 h in dark onice with 1) 10 μl of FITC-conjugated UIC2 (MDR-1) antibody (Santa CruzBiotechnology; SC-73354), 2) 1 μg of unconjugated UIC2 antibody (200μg/ml; Santa Cruz Biotechnology; SC-73354) or 3) 1 μg of unconjugatedisotype control (mouse IgG2a) antibody (eBioscience; Ref 14-4732-85).Cells labeled with unconjugated UIC2 and isotype control antibody werewashed twice with PBS containing 1% FCS, centrifuged and cell pelletlabeled with 5 μl (2.5 μg) of FITC-conjugated Goat F(ab)2 anti-mouse IgG(H+L) human adsorbed antibody (Southern Biotechnology; Cat #1032-02).Cells were washed twice with PBS containing 1% FCS and analyzed using BDverse flow cytometer. Cell fluorescence was analyzed in lymphoid gatebased on forward and side scatter, thereby focusing the analysis onperipheral blood lymphocytes (PBL).

When stained at 37° C. in the absence of P-glycoprotein inhibitorsreserpine and cyclosporine, 50% to 70% of lymphocytes were found to beTMRM-dull. In contrast, only a minor population of lymphocytes wereTMRM-dull when stained in the presence of reserpine and cyclosporine,suggesting that a reserpine- and cyclosporine-sensitive efflux pump isresponsible for the TMRM-dull phenotype of the vast majority oflymphocytes.

In addition to P-glycoprotein, a number of other ABC transporters havebeen described that are capable of pumping out lipophilic drugs,including dyes. To determine if the TMRM-dull phenotype of lymphocytescorrelates with P-glycoprotein expression, double staining with TMRM andFITC-conjugated UIC2 antibody was used. However, no correlation betweenTMRM staining and Pgp expression, as determined by staining withFITC-UIC2 was observed. The lack of correlation between TMRM stainingand FITC-UIC2 could be due to low level Pgp expression in lymphocyteswhich was not detected by FITC-conjugated UIC2 antibody. To increase thesensitivity of Pgp detection, the above experiment was repeated byperforming indirect immunofluorescence labeling with UIC2 antibody. Theuse of indirect labeling results in increased sensitivity as eachmolecule of UIC2 antibody can be potentially bound by many molecules ofFITC-labeled secondary antibody. Additionally, to further improve thesensitivity of Pgp expression, the amount of secondary antibody used wasincreased and the incubation volume was reduced, thereby resulting inhigh concentration of the secondary antibody. The incubation time wasalso increased to 2 h. Finally, to reduce non-specific binding of thesecondary antibody, F(ab)₂ fragment (instead of whole antibody) that hadbeen adsorbed against human serum proteins was used. Using thisoptimized staining protocol, robust expression of Pgp was observed inlymphocytes. More importantly, there was a strong inverse correlationbetween the levels of P-gp expression, as measured by UIC2 staining, andthe retention of TMRM in the lymphocytes, indicating that the dye effluxwas directly correlated with P-gp expression (FIG. 1F). Fluorescenceactivated cell sorting (FACS) analysis was also conducted on cells thathad been stained with TMRM in the presence of Pgp inhibitors (10 μMResperine and 2 μM cyclosporine) and then stained with UIC2 followed byFITC conjugated Goat antimouse IgG. A vast majority of Pgp-expressingcells stained brightly with TMRM under these conditions (FIG. 1H, 1I,1K, L). These results demonstrate that (i) Pgp expression has a majorinfluence on TMRM staining of PBL; and (ii) metabolic state, as measuredby mitochondrial membrane potential, is not the only determinant of TMRMstaining.

Example 2. Using Immunofluorescence Staining to Enrich for PgpExpressing Cells

Peripheral blood mononuclear cells (10 million cells) were stained asdescribed in the previous example except three monoclonal antibodiesagainst Pgp, including UIC2, MRK16, and 4E3 are used as primaryantibodies (each at a concentration of 0.5 μg/million cells) to increasethe sensitivity of the assay. Following extensive staining, cells werestained with FITC conjugated 5 μl (2.5 μg) of FITC-conjugated GoatF(ab)2 anti-mouse IgG (H+L) human adsorbed antibody (SouthernBiotechnology; Cat #1032-02). Cells were washed and free antibodybinding sites were blocked by addition of mouse IgG. After 2 washes,cells were labeled with PE-conjugated human CD8 antibody andAPC-conjugated CD4 antibody for 1 h at 4° C. Cells were analyzed by Flowcytometry and sorted into different fractions (e.g Pgp⁺, Pgp⁺/CD8⁺,Pgp⁺/CD4⁺, Pgp⁺/CD8⁻, Pgp⁺/CD4⁻, Pgp⁻/CD8⁺, Pgp⁻/CD4⁻).

Example 3. Using MACS (Magnetic Activated Cell Sorting) to Enrich forPgp Expressing Cells

Protocol 1. The following protocol was used to enrich Pgp expressingcells by MACS on staining with Pgp specific monoclonal antibodies andGoat anti-mouse IgG2a+IgG2b magnetic beads (Miltenyi). The blood samplesto isolate peripheral blood mononuclear cells (PBMCs) were obtained fromhealthy de-identified adult donors. PBMC were isolated from buffy coatsby Ficoll-Hypaque gradient centrifugation. Approximately 50 millionPBMCs growing in XVIVO medium supplemented with hIL2 were stained withTMRM (20 nM) for 30 min at 37° C. Cells were centrifuged and cell pelletblocked with 500 μl of human serum for 1 hour at 4° C. TMRM-stained 50million cells were separated into 3 tubes as follows: Tube 1: Stainedwith Pgp-UIC2 (unconjugated) antibody (1 μg/1 million cells) from SantaCruz Biotech; Tube 2: stained with an Pgp-4E3 antibody (1 μg/1 millioncells) from Abcam; Tube 3: stained with both UIC2 and 4E3 antibodieseach at 1 μg/1 million cells. Cells were incubated with the aboveantibodies for 2 hours at 4° C. 2 ml of MACS buffer was added to eachtube, cells were centrifuged at 300×g for 10 min at 4° C. andresuspended in 80 μl MACS buffer/tube. 20 μl of anti-Mouse IgG2a+bmicrobeads (Miltenyi: 130-047-201) were added to each tube and cellsincubated at 4° C. for 30 min. 2 ml of MACS buffer was added to eachtube, cells centrifuged and resuspended into 500 μl MACS buffer. Cellswere loaded cells on pre-washed MS columns (Miltenyi). Flow-throughfraction was collected as negative cell fraction and column-bound cellswere eluted as MDR1⁺ (Pgp⁺) cells following manufacturer's instructions.Aliquots of the positive and negative cells were analyzed by flowcytometer.

The results showed that there was enrichment for TMRM-dull cells(representing Pgp expressing cells) in the cells isolated based on UIC2and 4E3 staining alone. More importantly, there was greater enrichmentfor TMRM-dull cells among the cells isolated based on simultaneousstaining with both UIC2 and 4E3. The above results demonstrated thatTMRM-dull cells can be purified based on staining with Pgp-specificantibodies that bind to the extracellular domain of Pgp. Furthermore,combination of Pgp specific antibodies, particularly those that bind todifferent epitopes of Pgp, can be used to obtain higher yield andgreater purity. In addition to UIC2 and 4E3, a number of other Pgpantibodies are commercially available (e.g. MRK16, REA495) and can beused either alone or in combination to purify Pgp expressing (TMRM-dull)cells for the purpose of cellular therapies. Polyclonal antibodiesagainst the extracellular domain of Pgp can be also used for the purposeof this disclosure. A rabbit polyclonal against human MDR1 protein isavailable from Bioss Inc. Finally, other Pgp binding moieties, such asscFv, single domain antibodies, F(ab)2 fragments, affibodies, nanobodiesetc., can be used for the purpose of this invention. These antibodies(monoclonal and polyclonal) and antibody like moieties, singly or incombination, can be also used to deplete Pgp-expressing cells from astarting cell population for the purpose of cell therapies where it isdesirable to deplete Pgp-expressing cells, such as to reduce Graft vshost disease in patients undergoing allogeneic stem cell transplant.

Example 4. Purification of Pgp Expressing Cells from Peripheral BloodMononuclear Cells Using MACS

Protocol 2. To enhance the purity and yield of Pgp expressing cells, theprocedure in the preceding example is repeated using a cocktail ofmonoclonal antibodies against Pgp including UIC2, 4E3, REA495 and MRK16.Each antibody is used at 0.5 μg/million cells. Staining with primaryantibodies is carried out at 4° C. for 1 h and after extensive washesthe cells are incubated with 50 μl of anti-Mouse IgG beads/million cellsfor 2-4 hr with intermittent shaking. Positive and negative fractionsare isolated as described in the previous section. The modifiedprocedure is shown to result in greater yield and purity ofPgp-expressing cells.

Example 5. Purification of Pgp Expressing Cells from Peripheral BloodMononuclear Cells Using MACS

Protocol 3. Biotinylated REA495 antibody against Pgp and streptavidinmicrobeads were purchased from Miltenyi Biotech. PBMC or T cells islabeled with Biotinylated REA495 at 4° C. for 1 h followingmanufacturer's recommendation. After extensive washes with labelingbuffer (Miltenyi Biotech), the cells are resuspended in 90 μl oflabeling buffer per 10⁷ cells. Then, 40 μl of streptavidin microbeadsare added to the cells. Cells are mixed and refrigerated at 4-8° C. for1-2 hr with intermittent shaking. Cells are washed with 1-2 ml of bufferand centrifuged at 300 g for 10 min at 4-8° C. Cells are resuspended in500 μl of separation buffer and used for magnetic separation followingthe recommendations of the manufacturer. Positive and negative fractionsare isolated as described in the previous section.

Example 6. Purification of Pgp Expressing Cells from T Cells Using MACs

Protocol 4. In the preceding examples, Pgp expressing cells wereisolated from Peripheral blood mononuclear cells (PBMC) that wereobtained from Ficoll-Hypaque separation. To purify Pgp expressing Tcells, PBMC are enriched for T cells using a Pan T cell Isolation kit(Catalog #130-096-535) available from Miltenyi and following themanufacturer's recommendations. This kit uses a cocktail of antibodiesagainst markers that are not present on T cells to deplete cellsbelonging to other lineages. Similar kits are available from othersources as well. The T cell enriched fraction is then positivelyselected for Pgp expressing cells using the protocol 1, 2 or protocol 3described above.

Example 7. Purification of Pgp Expressing Cells from PBMC UsingPhotodynamic Cell Therapy with TH9402

T cells were isolated using PAN T-cell isolation kit (Miltenyi cat no.130-096-535) from buffy coat preparation following Ficoll-Hypaqueseparation and RBC lysis as described above. 12 million T cells wereresuspended at 1 million cells/ml in XVIVO T cell medium (Lonza)supplemented with 5 ng/ml IL7. Half (6 million) of the T cells were leftuntreated while the remaining half were treated with 10 μM of TH9402compound. Cells were incubated at 37° C. in a water bath in dark for 40min, washed with T-cell medium and then resuspended in TH9402-freeT-cell medium. Cells were allowed to efflux TH9402 at 37° C. in dark in10 ml of T cell medium for 2 h. Cells were centrifuged and re-suspendedin fresh medium. Each sample was then plated in 2 wells of two different6-well plates. One plate was left unexposed to light and the secondplate was exposed for 1 h to light (1000×10 Lux units) from an LED lamp.Alternatively, light treatment can be achieved by exposure to afluorescent light-scanning device (PDCT-Xerox Series 4,Theratechnologies) delivering 5 J/cm² at wavelength of 514 nm. Afterlight exposure, cells were incubated in the XVIVO T cell medium with 5ng/ml IL7 at 37° C. for 2 days in a 5% CO₂ incubator. After 2 days, analiquot of the cells were stained at 4° C. for 40 min with DiOC2(3) (60ng/ml) in 10 ml RPMI with 10% FBS medium. DiOC2(3) is a known substrateof Pgp. Cells were centrifuged, washed, resuspended in 10 ml of dye-freeRPMI-10% FBS medium to efflux dye at 37° C. for 90 min. After Dyeefflux, the cells were stained with Propidium iodide (PI) and examinedby flow cytometry. The percentage of cells in the lymphoid gate in thevarious treatment groups are shown in the following table. The resultsshow that even in the group which was untreated with TH904, there issignificant enrichment for P-glycoprotein expressing lymphoid cells thatstain dull with DiOC2(3) upon exposure to light (12% vs 62%), suggestingthat light exposure, by itself, can lead to enrichment of Pgp-expressingT lymphocytes. Furthermore, in the group that was treated with theTH9402 compound and then exposed to light, there was further enrichmentfor Pgp-expressing T cells (from 10% to 80%).

Essentially similar results were obtained when the experiment wasrepeated with PBMC rather than purified T cells.

% live % Pgp⁺ cells (DiOC2 Sample cells (P1) (3)-dull) T-Untreated (UT)-75 12 unexposed T-TH9402-treated- 78 10 unexposed T-UT-1 h exposure to21 62 light T-TH9402-1 h exposure to 10 80 light

After 6 days, 250 μl cell aliquot from T cells were stained withDiOC2(3), allowed to efflux the dye and then stained with CD62L-APC, amarker present on human memory stem T cells (T_(SCM)) with stem likeproperties.

% Pgp⁺ % Pgp⁺ (DiOC2 (3)- (DiOC2(3)- dull cells dull) (based on dyeCD62L+ efflux) (gated cells % live on live (gated on Sample cells (P1)cells) live cells) T-UT-unexposed 77 5 3 T-TH-unexposed 75 4 2 T-UT-1 hexposure 24 6 3 to light T-TH9402-1 h 14 70 69 exposure to light

After 9 days, 1 ml cell aliquot from T cells were stained with DiOC2(3),allowed to efflux the dye and then stained with CD62L-APC, a markerpresent on human memory stem T cells (T_(SCM)) with stem likeproperties. Cells were then analyzed by flow cytometry. The % of cellsin the different fractions are shown in FIG. 2 and the following tablewhich show there is a significant decline in cell viability followingexposure to light in both TH9402-treated and untreated cells. However,among the live cells that had been treated with TH9402 and then exposedto light, there is a significant enrichment (from 45% to 79%) for cellsthat express Pgp (i.e. that are DiOC2(3)-dull) as compared to cells thathad not been treated with TH9402 but were then exposed to light.Furthermore, among the live cells that had been treated with TH9402 andthen exposed to light, there is a significant enrichment (from 21% to67%) for cells that express both Pgp (i.e. that are DiOC2(3)-dull) andCD62L as compared to cells that had not been treated with TH9402 butwere then exposed to light.

% Pgp⁺ % Pgp⁺ (DiOC2 (3)- (DiOC2 (3)- % live dull cells dull), cells(Based on CD62L+ (Propidium dye efflux) cells iodide (gated on (gated onlive Sample negative) live cells) cells) T-UT-unexposed 75 46 19T-TH9402-unexposed 79 45 21 T-UT-1 h exposure to 38 50 49 lightT-TH9402-1 h exposure 27 79 67 to light

Example 9. Sorting of Pgp⁺ Cells, Expression of CAR in Sorted Cells

T Cells were isolated form donor blood. PBMC were isoalted after RBClysis and Ficoll gradient. 130 million T cells were isolated from 400million PBMCs using T-PAN isolation kit, cat no. 130-096-535 fromMiltenyi. T cells were cultured in XVIVO medium with IL2 100 IU/ml. CD3or CD28 antibodies were not added.

100 million T cells were washed with PBS+1% hAB serum at 4° C. andblocked with 500 μl hAB serum at 4° C. for 1 h followed by washing (2times). Mixture of two primary antibodies to stain (Pgp⁺) MDR+ cellswere used: MDR (UIC2) sc-73354 0.5 μg per million cells andP-glycoprotein antibody from Abcam, Ab 10333 0.2 μg per million cells,were used and incubated at 4° C. for 1 h followed by washing twice at 4°C.

Secondary antibody Goat F(ab′)₂-a-mouse-IgG(H+L) human ads-FITC cat no1032-02, Southern Biotech is prepared as a stock 0.5 mg/ml. For 100million T cells, 250 μl of secondary antibody was added to cell pelletsre-suspended in residual wash buffer, no extra buffer added. The pelletswere stained for 2 hours, with intermittent shaking at 4° C., followedwith two washings. The cells were re-suspended in PBS with 1 μg/mlPropidium Iodide (PI) to exclude dead cells. Cells were checked on flowfor FITC staining before sorting.

Cells were sorted on a MoFlo machine. Dead, PI+, cells were excluded andFITC⁺ and FITC⁻ cells collected. 9 million FITC⁺ cells and 7.5 millionFITC⁻ cells were collected, cultured in 6 well plates with complete Tcells medium supplemented with IL2 (100 IU/ml), and CD3 (30 ng/ml) andCD28 (30 ng/ml) antibodies.

Pgp⁺ and Pgp⁻ cells were infected with FMC63-BBz-A13 CAR virus, byinfection three times on three days using Polybrene at 18 μl per well.Spinfection was performed at 2800 rpm, 32° C. for 90 min, and the mediumwas replaced with fresh medium after 6 h of infection.

Cells were expanded without selection with puromycin in IL2, CD3, andCD28 containing T cell medium. MDR⁺ sorted cells formed bigger clumps ascompared to MDR⁻ cells. Thus showing that Pgp⁺ cells proliferate morecompared to Pgp⁻ cells

Total cell Total cell Total cell Sample count-4 days count-6 dayscount-8 days Pgp⁺ sorted-FMC63- 7 million 12.2 million 12 millionBBz-A13 Pgp⁻ sorted FMC63- 1.26 million 0.42 million 0 million BBz-A13cells

Example 9. Purification of Pgp Expressing Cells by ChemotherapeuticSelection Criteria

T cells were isolated using CD3 microbeads. Cells were resuspended in Tcell culture medium and treated under the following conditions.

1. High dose Vincristine Treatment: Cells were treated with Vincristine(Sigma) at doses of 100, 250, 500, 750, 1000 ng/ml for 24 h. Thefollowing day, the medium was changed and cells cultured inVincristine-free medium.

2. Intermediate Dose Vincristine Treatment: Cells were treated withvincristine at intermediate doses of 5, 10, 20, 30, 50 ng/ml withcontinuous exposure of the drug thereafter.

3. Low dose Vincristine Treatment: Cells were treated with vincristineat low doses 0.5, 1, 2, 2.5, 3 ng/ml with continuous exposure to thedrug thereafter.

4. Cells were treated with Akt inhibitor VIII (Cat #124018, Calbiochem),at 1 μM final conc.

5. Cells were treated with Etoposide at dose 100, 500, 1000

6. Cells were treated with Adriamycin (doxorubicin) at 0.1, 0.5, 1 μg/ml

T cells treated with intermediate and high dose of vincristine werechecked for Pgp⁺ enrichment by DiOC2(3) efflux assay and cell death byPropidium iodide staining after 2 days of treatment. Cells were analyzedby flow cytometry. The results as shown in the table below demonstrate amodest enrichment of Pgp⁺ cells with higher dose vincristine treatmentsafter 2 days.

% Pgp⁺ (DiOC2(3)- Samples: 2 days post- % Cell Death (PI+ dull) (out oflive treatment cells) cells) Untreated 14 8 Vincristine-5 ng/ml 13 8Vincristine-10 ng/ml 15 9 Vincristine-20 ng/ml 15 9 Vincristine-30 ng/ml20 11 Vincristine-50 ng/ml 15 10 Vincristine-100 ng/ml 28 11Vincristine-250 ng/ml 31 11 Vincristine-500 ng/ml 27 13 Vincristine-750ng/ml 30 14 Vincristine-1 μg/ml 31 14

T cells treated with low, intermediate and high dose of vincristine werechecked for Pgp⁺ enrichment by DiOC2(3) efflux assay and cell death byPropidium iodide staining after 2 days of treatment and after 7 days oftreatment. The results are shown in the following table and demonstratethat after 7 days of vincristine treatment, there was a significantenrichment of Pp cells with 1 μg/ml Vincristine from 5% in untreatedgroup to 18% in the 1 μg/ml-treated group.

% Pgp+ (DiOC2(3)- Samples: 7 days post- % Cell Death (PI+ dull) (gatedon live treatment cells) cells) UT 9 5 Vincristine-0.5 ng/ml 10 7Vincristine-1 ng/ml 12 7 Vincristine-2 ng/ml 8 7 Vincristine-2.5 ng/ml10 7 Vincristine-3 ng/ml 14 9 Vincristine-5 ng/ml 9 8 Vincristine-10ng/ml 9 10 Vincristine-20 ng/ml 9 10 Vincristine-50 ng/ml 14 9Vincristine-100 ng/ml 16 13 Vincristine-250 ng/ml 25 11 Vincristine-500ng/ml 26 9 Vincristine-750 ng/ml 29 10 Vincristine-1 μg/ml 23 18

T cells treated with drugs other than vincristine were checked for Pgp⁺enrichment after 8 days of treatment using the assay described above.Treatment with adrimycin (doxorubicin) at 1 μg/ml showed significantenrichment of Pgp⁺ or DiOC2(3)-dull cells.

Sample (8 days post- % Cell Death (PI+ % pgp+ (out of live treatment)cells) cells) UT 3 16 Rapamycin-100 ng/ml 11 15 RAD1001-0.2 ng/ml 7 15RAD1001-1 ng/ml 5 15 Adriamycin-0.1 μg/ml 5 16 Adriamycin-0.5 μg/ml 7 16Adriamycin-1 μg/ml 12 27 Etoposide-0.1 uM 5 15 Etoposide-0.5 unM 4 16Etoposide-1 uM 5 15 Akt-inhibitor-1 uM 4 19 Verapamil-1 uM 4 17Verapamil-5 uM 3 17 Verapamil-10 uM 3 17

Example 10. Temperature Selection of Pgp-Expression Cells

T cells were isolated as described above. Three different water bathswere set up at 42, 43 and 44° C. Four 6-well plates labelled as 37, 42,43 and 44 containing 1 ml of T-cell medium were kept at 37° C. incubatorto pre-warm the media. T cells (1 million cells/ml) in 1 ml of T-cellmedium with IL7 (5 ng/ml) were kept at 37° C. or in the above waterbaths for the indicated time intervals as follows:

1. 37° C.

2. 42° C. for 1 h

3. 42° C. for 2 h

4. 42° C. for 3 h

5. 42° C. for 4 h

6. 42° C. for 5 h

7. 42° C. for 6 h

8. 43° C. for 1 h

9. 43° C. for 2 h

10. 43° C. for 3 h

11. 43° C. for 4 h

12. 43° C. for 5 h

13. 43° C. for 6 h

14. 44° C. for 1 h

15. 44° C. for 2 h

16. 44° C. for 3 h

17. 44° C. for 4 h

18. 44° C. for 5 h

19. 44° C. for 6 h

After each time point, cells were added to separate wells of 6 wellplates containing the pre-warmed medium and kept at 37° C. for rest ofthe experiment.

After 5 days the percentage of Pgp⁺ cells were checked by DiOC2(3)efflux to check if exposure of T cells to high temperature for shorttime point can enrich Pgp⁺ cell population. The results are shown in thefollowing Table and demonstrate significant enrichment of Pgp⁺(DiOC2(3)-dull) cells following exposure to elevated temperatures. Forexample, DiOC2(3)-dull cells showed enrichment from 76% to 98% whenexposed to 43° C. for 2 hours as compared to cells kept at 37° C.

Pgp+ (DIOC2- effluxing cells (gated on live Sample Live cells (%) cells)T-37° C. 65 76 T-42° C.-1 h 62 77 T-42° C.-2 h 67 96 T-42° C.-3 h 50 93T-43° C.-1 h 60 93 T-43° C.-2 h 20 98 T-43° C.-3 h 4 95

Peripheral blood stem cell cells were obtained from a patient undergoingstem cell transplantation. Cells underwent RBC lysis to get rid of redcells and Ficoll-Hypaque separation to enrich for mononuclear cells.Approximately, 10 million cells were recovered and approximately 7million cells were used for hyperthermia experiment. Cells wereresuspended in 15 ml Falcon tubes at approximately 1 million cells/mland were kept at 37° C. or in a water-bath at 43° C. for the indicatedtime intervals as follows:

37° C.

43° C. for 0.5 h

43° C. for 1 h

43° C. for 1.5 h

43° C. for 2 h

43° C. for 2.5 h

43° C. for 3 h

Following exposure cells were transferred to a 6 well plate at incubatedat 37° C. in Stem-cell medium XVIVO-10 supplemented with SCF, TPO, FLT3,IL3, IL6 (all at 50 ng/ml) in a humidified 5% CO₂ incubated for 72 h.100 μl aliquots were then stained with 1 μg/ml Propidium iodide to checkcell death.

% lymphocytes (P1 % PI+ve Sample population) dead cells T-37° C. 18 12T-43° C.-0.5 h 16 13 T-43° C.-1 h 8 23 T-43° C.-1.5 h 6 31 T-43° C.-2 h7 29 T-43° C.-2.5 h 5 39 T-43° C.-3 h 5 36

After 96 hours, cells were stained with DiOC2(3) (60 ng/ml in 5 ml ofRPMI 10% FBS medium at 4° C. for 40 min). The cells were washed withmedium, dye-efflux in 10 ml RPMi 10% medium at 37° C. for 90 min, washedtwice with PBS 1% FBS, and stained with 1.5 μl/sample/100 μl ofCD34-APCefluor 780 (ebiosciences cat #470349-42) at 4° C. for 1 h. Thecells were washed and analyzed by Flow Cytometry. APC-efluor-780 wasdetected in APC-Cy7 channel in BD Facsverse. The results demonstratesignificant enrichment of Pgp⁺ cells from 48% to 76-80% followingexposure to 43° C. for different time intervals. In addition there isenrichment of Pgp⁺/CD34⁺ stem cells from 1% to 2% in cells exposed to43° C. for 3 h as compared to cells cultured at 37° C.

% Pgp⁺ (DiOC2- Sample effluxing cells) % Pgp⁺CD34⁺ T-37° C. 48 1 T-43°C.-0.5 h 76 0.3 T-43° C.-1 h 70 1 T-43° C.-1.5 h 76 0.5 T-43° C.-2 h 761 T-43° C.-2.5 h 76 0.5 T-43° C.-3 h 80 2

Example 11. Use of Pgp Enriched Cells for Adoptive Cellular Therapy

The blood samples to isolate peripheral blood mononuclear cells (PBMCs)are obtained from healthy de-identified adult donors. PBMC are isolatedfrom buffy coats by Ficoll-Hypaque gradient centrifugation. Pgpexpressing cells are purified from PBMC by any one or more of themethods described in the previous examples, including Flow sorting, MACSand photodynamic cell therapy with TH9402, selection with vincristineand hyperthermia. Pgp⁺ T cells, Pgp⁻ T cells and unpurified T cells arere-suspended in XVIVO medium (Lonza) supplanted with 10 ng/ml solubleanti-CD3, 10 ng/ml soluble anti-CD28 and 100 IU recombinant human-IL2.Cells are engineered to express FMC63(vL-vH)-Myc-BBz-PAC ChimericAntigen Receptor (CAR) targeting human CD19 by infection withpLENTI-EF1a-FMC63(vL-vH)-Myc-BBz-T2A-Pac-A13 lentiviral vector. NSG mice(Jackson Lab) are sub-lethally irradiated at a dose of 175 cGy. 24 hourspost irradiation (day 2), mice are injected with 2.5×10⁴ RAJI cells viatail-vein. On day 3, the mice (n=5 for each group) are injected by tailvein with 1 million Pgp⁺, Pgp⁻, or unpurified T cells that had beeninfected with the FMC63(vL-vH)-Myc-BBz-PAC lentivirus. Control mice(n=5) are injected with RAJI cells but do not receive T cells. Survivalof mice injected with Pgp⁺ CAR-T cells is significantly higher thanthose of mice injected with Pgp⁻ CAR-T cells or unpurified T cells. Thisis true irrespective of the method (flow sorting, MACS or TH9402 pluslight exposure, selection with vincristine or hyperthermia) used topurify Pgp⁺ cells. PCR analysis for the presence of CAR-modified T cellsin blood and bone marrow reveals longer in vivo persistence of CAR-Tcells that are derived from Pgp⁺ cells.

The above experiment is repeated using Pgp⁺/CD8⁺, Pgp⁻/CD8⁺, Pgp⁺/CD4⁺,Pgp⁺/CD4⁻ starting population of cells. Again, CAR generated from Pgp+vecells perform better than those generated from Pgp⁻ cells and persistedlonger in vivo.

Example 12. Use of Autologous Pgp-Expressing Cells for Adoptive CellTherapy

Patients with relapsed Acute lymphocytic Leukemia (ALL) or high-riskintermediate grade B-cell lymphomas may receive immunotherapy withadoptively transferred autologous Pgp⁺ T cells-derived CAR-T cells. Aleukapheresis product collected from each patient undergoes selection ofPgp⁺ T cells using Flow sorting with Pgp antibodies, MACS using Pgpantibodies, Photodynamic selection following exposure to TH9402 pluslight, selection with vincristine or hyperthermia. Cells are transducedwith clinical grade CD19CAR virus and then selection and expansion ofthe CAR-T cells occur in a closed system. After the resulting cellproducts have undergone quality control testing (including sterility andtumor specific cytotoxicity tests), they are cryopreserved. Meanwhile,following leukapheresis, study participants commence withlymphodepletive chemotherapy following which they receive theircryopreserved CAR-T cells. The CAR-T cell product is transported, thawedand infused at the patient's bedside. The dose of CAR-T product variesfrom 1×10⁴ CAR⁺ CD3 cells/kg to 1×10⁹ CAR⁺ CD3 cells/kg as per the studyprotocol. The CAR product may be administered in a single infusion orsplit infusions. Research participants can be pre-medicated at least 30minutes prior to T cell infusion with 15 mg/kg of acetaminophen P.O.(max. 650 mg) and diphenhydramine 0.5-1 mg/kg I.V. (max dose 50 mg).Clinical and laboratory correlative follow-up studies can then beperformed at the physician's discretion, and may include quantitativeRT-PCR studies for the presence of CD19-expressing ALL/lymphoma cellsand/or the adoptively transferred T cells; FDG-PET and/or CT scans; bonemarrow examination for disease specific pathologic evaluation; lymphnode biopsy; and/or long-term follow up per the guidelines set forth bythe FDA's Biologic Response Modifiers Advisory Committee that apply togene transfer studies.

Example 13. Use of Allogeneic Pgp-Expressing Cells for Adoptive CellsTherapy

Patients with relapsed Acute Lymphocytic Leukemia (ALL) or high-riskintermediate grade B-cell lymphomas who have undergone an allogeneicbone marrow transplant may receive immunotherapy with adoptivelytransferred allogeneic Pgp⁺ T cells-derived CAR-T cells. A leukapheresisproduct collected from the donor (same donor as used for the allogeneictransplant) undergoes selection of Pgp⁺ T cells using Flow sortingfollowing staining with Pgp antibodies, MACS following staining with Pgpantibodies, Photodynamic selection following exposure to TH9402 pluslight, selection with vincristine or hyperthermia. Cells are transducedwith clinical grade CD19-CAR and then selection and expansion of theCAR-T cells occur in a closed system. After the resulting cell productshave undergone quality control testing (including sterility and tumorspecific cytotoxicity tests), they are cryopreserved. Meanwhile, studyparticipants commence with lymphodepletive chemotherapy following whichthey receive the cryopreserved allogeneic CAR-T cells. The CAR-T cellproduct is transported, thawed and infused at the patient's bedside. Thedose of CAR-T product may vary from 1×10⁴ CAR⁺ CD3 cells/kg to 1×10⁹CAR⁺ CD3 cells/kg as per the study protocol. The CAR product may beadministered in a single infusion or split infusions. Researchparticipants can be pre-medicated at least 30 minutes prior to T cellinfusion with 15 mg/kg of acetaminophen P.O. (max. 650 mg) anddiphenhydramine 0.5-1 mg/kg I.V. (max dose 50 mg). Clinical andlaboratory correlative follow-up studies can then be performed at thephysician's discretion, and may include quantitative RT-PCR studies forthe presence of CD19-expressing ALL/lymphoma cells and/or the adoptivelytransferred T cells; FDG-PET and/or CT scans; bone marrow examinationfor disease specific pathologic evaluation; lymph node biopsy; and/orlong-term follow up per the guidelines set forth by the FDA's BiologicResponse Modifiers Advisory Committee that apply to gene transferstudies. Use of immunosuppressive drugs is also at the discretion of thephysician.

Example 14. Use of Pgp Negative T Cells to Reduce the Incidence of GVHDin Patients Undergoing Allogeneic Bone Marrow Transplant and OtherDisorders

Peripheral blood stem cells are obtained from a donor usingleukopheresis following standard procedures. The donor may be, forexample, an HLA-matched (10/10 match) sibling donor, 10/10 matchedunrelated donor, or Antigen mismatched sibling or unrelated donor, or ahaploidentical donor. The leukopheresed product is enriched forCD34-expressing cells by positive selection using the CliniMACS Prodigy®System from Miltenyi Biotec and following the manufacturer'srecommendations. The CD34⁻ fraction is labeled with one or moreantibodies against extracellular domain of Pgp (e.g. UIC2, 4E3, MRK16etc.) followed by incubation with Goat anti-mouse IgG magnetic beads andnegative selection using the CliniMACS Prodigy® System, and followingthe manufacturer's recommendations. Alternatively, negative selectionfor Pgp-expressing cells can be obtained by using one, or a cocktail, ofPgp antibodies that are directly conjugated to magnetic beads. The Pgpnegative fraction should contain less than 50% Pgp-expressing cells, orpreferably less than 40% Pgp-expressing cells, or preferably less than30% Pgp-expressing cells, or preferably less than 20% Pgp-expressingcells, or preferably less than 10% Pgp-expressing cells, or preferablyless than 5% Pgp-expressing cells, or preferably less than 1%Pgp-expressing cells. The patient who has received the conditioningregimen (myeloablative or reduced intensity or non-myeloablative) toprepare for transplant is then administered by intravenous infusion theCD34 enriched stem cell fraction along with Pgp-depleted T cellfraction. The proportion and amount of Pgp-depleted T cell fraction thatis administered to the patient is at the discretion of the physician.For example, from 1×10⁴ CD3 cells/kg to 1×10⁹ CD3 cells/kg may beinfused either as a single infusion or split infusion depending on thetolerance of the patient and discretion of the treating physician.

Example 15. Use of Pgp-Negative T Cell Product for the Treatment of CMV(Cytomegalovirus) Infection in an Allogeneic Stem Cell TransplantRecipient

A patient who is status-post allogeneic stem cell transplant from anunrelated donor develops refractory CMV infection. Peripheral bloodmononuclear cells are collected from the original donor, who is CMVseropositive. T cells are first enriched by negative selection using acocktail of antibodies against non-T cell markers and using theCliniMACS Prodigy® System. The T cell fraction is then depleted ofPgp-expressing cells by incubation with a Pgp antibody or cocktail ofantibodies (1-2 μg/million cells) followed by negative selection usingthe CliniMACS Prodigy® System, and following the manufacturer'srecommendations. The Pgp-depleted fraction of T cells is administered tothe patient intravenously either as a single infusion or in increasingfractions, at the discretion of the treating physician. For example,from 1×10⁴ Pgp⁻/CD3⁺ cells/kg to 1×10⁹ Pgp⁻/CD3⁺ cells/kg may be infusedeither as a single infusion or split infusion depending on the toleranceof the patient and discretion of the treating physician.

Example 16. Use of Pgp-Negative T Cell Product for the Treatment of CMV(Cytomegalovirus) Infection in an Immunodeficient HIV/AIDS Recipient

A patient with HIV/AIDS develops refractory CMV infection. Peripheralblood mononuclear cells are collected from an HLA matched donor, who isCMV seropositive. T cells are first enriched by negative selection usinga cocktail of antibodies against non-T cell markers and using theCliniMACS Prodigy® System. The T cell enriched fraction is then depletedof Pgp-expressing cells by incubation with a Pgp antibody or a cocktailof antibodies (1-2 μg/million cells) followed by negative selectionusing the CliniMACS Prodigy® System, and following the manufacturer'srecommendations. The Pgp-depleted fraction of T cells is administered tothe patient intravenously either as a single infusion or in increasingfractions, at the discretion of the treating physician. For example,from 1×10⁴ Pgp⁻/CD3⁺ cells/kg to 1×10⁹ Pgp⁻/CD3⁺ cells/kg may be infusedeither as a single infusion or split infusion depending on the toleranceof the patient and discretion of the treating physician.

Example 17. Use of Pgp-Negative T Cell Product for the Treatment ofAdenovirus Infection in an Allogeneic Stem Cell Transplant Recipient

A patient who is status-post allogeneic stem cell transplant from anunrelated donor develops refractory adenovirus infection. Peripheralblood mononuclear cells are collected from the original donor, who isadenovirus seropositive. T cells are first enriched by negativeselection using a cocktail of antibodies against non-T cell markers andusing the CliniMACS Prodigy® System. The T cell fraction is thendepleted of Pgp-expressing cells by incubation with a Pgp antibody orcocktail of antibodies (1-2 μg/million cells) followed by negativeselection using the CliniMACS Prodigy® System, and following themanufacturer's recommendations. The Pgp-depleted fraction of T cells isadministered to the patient intravenously either as a single infusion orin increasing fractions, at the discretion of the treating physician.For example, from 1×10⁴ Pgp⁻/CD3⁺ cells/kg to 1×10⁹ Pgp⁻/CD3⁺ cells/kgmay be infused either as a single infusion or split infusions dependingon the tolerance of the patient and discretion of the treatingphysician.

Example 18. Use of Pgp-Negative T Cell Product for the Treatment of BKVirus Infection in an Allogeneic Stem Cell Transplant Recipient

A patient who is status-post allogeneic stem cell transplant from anunrelated donor develops refractory BK virus infection. Peripheral bloodmononuclear cells are collected from the original donor BK virusseropositive. T cells are first enriched by negative selection using acocktail of antibodies against non-T cell markers and using theCliniMACS Prodigy® System. The T cell fraction is then depleted ofPgp-expressing cells by incubation with a Pgp antibody or cocktail ofantibodies (1-2 μg/million cells) followed by negative selection usingthe CliniMACS Prodigy® System, and following the manufacturer'srecommendations. The Pgp-depleted fraction of T cells is administered tothe patient intravenously either as a single infusion or in increasingfractions, at the discretion of the treating physician. For example,from 1×10⁴ Pgp⁻/CD3⁺ cells/kg to 1×10⁹ Pgp⁻/CD3⁺ cells/kg may be infusedeither as a single infusion or split infusion depending on the toleranceof the patient and discretion of the treating physician.

Example 19. Enrichment of Pgp⁺/CD34⁺ Pluripotent Hematopoietic StemCells Using Serum-Starvation

The most primitive pluripotent hematopoietic stem cells are known toco-express CD34 and Pgp and efflux DiOC2(3) (Chaudhary and Roninson,Cell, 66, 85-94, 1991). To examine if these cells can be enriched byserum starvation, peripheral blood stem cell cells were collected byapheresis from a patient undergoing stem cell transplantation aftermobilization with chemotherapy and G-CSF using standard procedures forstem cell mobilization and collection. Cells underwent RBC lysis to getrid of red cells and Ficoll-Hypaque separation to enrich for mononuclearcells. Cells were cultured for 6 days in RPMI medium containing 2%, 5%or 10% Fetal bovine Serum (FBS). Cells were stained with DiOC2(3) (60ng/ml in 5 ml of RPMI 10% FBS medium at 4° C. for 40 min). The cellswere washed with medium, dye-efflux in 10 ml RPMi 10% medium at 37° C.for 90 min, washed twice with PBS 1% FBS, and stained with 1.5μl/sample/100 μl of CD34-APCefluor 780 (ebiosciences cat #470349-42) at4° C. for 1 h. The cells were washed and analyzed by Flow Cytometry.APC-efluor-780 was detected in APC-Cy7 channel in BD Facsverse. FIG. 3shows significant enrichment of CD34⁺ stem cells from 4.6% to 5% to 7%upon reduction of FBS from 10% to 5% to 2%. Furthermore, there wassignificant enrichment of the most primitive hematopoietic stem cellsthat are DiOC2(3)-dull (or Pgp⁺) and CD34⁺ from 0.6% to 2% to 4% uponreduction of FBS from 10% to 5% to 2%. These results demonstrate thatCD34⁺ hematopoietic stem cells and CD34⁺/Pgp⁺ pluripotent hematopoieticstem cells can be enriched by serum starvation.

Example 20. Enrichment of Pgp⁺/CD34⁺ Pluripotent Hematopoietic StemCells by Growth Factor-Starvation

The experiment was conducted as in the preceding example except thatcells were cultured for 6 days in XVIVO-10 (Lonza) medium alone orXVIVO-10 medium containing SCF (50 ng/ml), TPO (50 ng/ml), FLT3 (50ng/ml), IL3 (50 ng/ml) and IL6 (50 ng/ml). There was an enrichment ofCD34⁺/DiOC2(3)-dull pluripotent hematopoietic stem cells from 2% to 4%when the cells were cultured in the medium lacking growth factors (FIG.4 ).

Example 21. Use of Pgp-Positive/CD34⁺ Cell Product for Allogeneic StemCell Transplantation

A patient with aplastic anemia is a candidate for allogeneic bone marrow(or peripheral blood stem cell) transplant from a one antigen mismatchedrelated donor. Bone marrow is harvested from the donor under generalanesthesia. In order to reduce the incidence of graft-vs-host disease,the bone marrow is enriched for hematopoietic stem cells by positiveselection for CD34⁺ cells using CliniMACS Prodigy® System. To furtherenrich for the most primitive hematopoietic progenitors, the CD34⁺ cellfraction is enriched for cells that are Pgp⁺ by exposure to TH9402 pluslight, selection with vincristine or hyperthermia. For enrichment usingTH9402, cells are incubated at 37° C. with 10 μM TH9402(Theratechnologies, Montreal, QC, Canada) in X-Vivo 15 medium with 2.5%HAB. After a 40-minute incubation, cells are centrifuged and dye effluxfavored by resuspending cells in TH9402-free medium for 90 minutes. Atthe end of the latter dye efflux period, cells are exposed to afluorescent light-scanning device (PDCT-Xerox Series 4,Theratechnologies) delivering 5 J/cm² at a wavelength of 514 nm. Forenrichment using hyperthermia, the cells are exposed to 43° C. for 3hours. The final CD34⁺/Pgp⁺ cellular product (2.5-5 million cells/Kg ofbody weight) is used for allogeneic transplantation after the patienthas received myeloablative conditioning regimen. Patient receivesstandard post-allogeneic transplant care, including use ofimmunosuppressive drugs, under the direction of the treating physician.

Example 22. Use of Pgp-Positive/CD34+ Cell Product for Gene Therapy

A patient with X-linked severe combined immunodeficiency (SCID-X1) is acandidate for gene therapy with interleukin-2 receptor γ-chain (γc)complementary DNA. Bone marrow is harvested from the patient undergeneral anesthesia. Bone marrow is enriched for hematopoietic stem cellsby positive selection for CD34⁺ cells using CliniMACS Prodigy® System.To further enrich for the most primitive hematopoietic progenitors, theCD34⁺ cell fraction is enriched for cells that are Pgp⁺ by exposure toTH9402 plus light, selection with vincristine or hyperthermia. Forenrichment using TH9402, cells are incubated at 37° C. with 10 μM TH9402(Theratechnologies, Montreal, QC, Canada) in X-Vivo 15 medium with 2.5%HAB. After a 40-minute incubation, cells are centrifuged and dye effluxfavored by resuspending cells in TH9402-free medium for 90 minutes. Atthe end of the latter dye efflux period, cells are exposed to afluorescent light-scanning device (PDCT-Xerox Series 4,Theratechnologies) delivering 5 J/cm² at a wavelength of 514 nm. Forenrichment using hyperthermia, the cells are exposed to 42.5° C. for 3hours. The final CD34⁺/Pgp⁺ cellular product (5 million cells/Kg of bodyweight) is used for gene transfer with a lentiviral vector encoding theγc cDNA using published regimen (Gaspar H B et al., Lancet, 2004;364:2181-7). Patient receives infusion of approximately 5×10⁶/KgCD34⁺/Pgp⁺ gene modified stem cells without preparative conditioning.Patient showed sustained recovery of T cells, including CD3⁺, CD8⁺ andCD4⁺ subsets and normal immunological function.

Example 23. Enrichment of Stem Like T Cells Using Serum-Starvation

To examine if T stem cells can be enriched by serum starvation, theexperiment in example 19 is repeated using T cells isolated fromperipheral blood. There is enrichment of the DiOC2(3)-dull (or Pgp+)cells following culture in RPMI medium containing 2% FCS as compared toRPMI medium containing 10% serum. These results demonstrate that T cellswith stem like characteristic and/or Pgp⁺ T cells can be enriched byserum starvation.

A number of embodiments have been set forth above to illustrate theinvention. The following claims further set forth what the Applicantsregard as their invention.

What is claimed is:
 1. A method for isolating T cells and/or NK cellssuitable for adoptive cell therapy, comprising: (a) obtaining a samplecomprising T cells and/or NK cells; (b) optionally enriching the samplefor T cells and/or NK cells; and (c) isolating p-glycoprotein positive(Pgp+) T cells and/or NK from the sample, so as to obtain a fractionenriched in Pgp-positive T cells and/or NK cells by (i) contacting thesample with at least one phototoxic compound that is a substrate of Pgp;and (ii) exposing the sample to a light source sufficient to activatethe at least one phototoxic compound so as to kill Pgp-negative cells,thereby isolating T cells and/or NK cells suitable for adoptive celltransfer therapy.
 2. The method of claim 1, wherein the cells arecontacted with the at least one phototoxic compound for a time and at: atemperature sufficient to allow efflux of the phototoxic compound. 3.The method of claim 1, wherein after contact with the at least onephototoxic compound the cells are removed from the photototoxic compoundfor a time and at a temperature sufficient to allow efflux of thephototoxic compound.
 4. The method of claim 2, wherein the temperatureis 37° C.
 5. The method of claim 1, wherein the at least one phototoxiccompound is any one or more of2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid methylester hydrochloride,2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid ethyl esterhydrochloride, 2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoicacid octyl ester hydrochloride,2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid n-butylester hydrochloride, 2-(6-ethyl amino-3-ethylimino-3H-xanthen-9-yl)-benzoic acid n-butyl ester hydrochloride, orderivatives thereof or combinations thereof.
 6. The method of claim 1,wherein the light source is a visible light source.
 7. The method ofclaim 1, wherein the Pgp+ cells are further enriched using other methodsfor Pgp enrichment.
 8. The method of claim 1, wherein the fractionenriched in Pgp+ cells contains less than (a) 50% Pgp− cells; b) 40%Pgp− cells; c) 30% Pgp− cells; d) 20% Pgp− cells; d) 10% Pgp− cells; e)5% Pgp− cells; or f) 1% Pgp-n cells.
 9. The method of claim 1, whereinthe Pgp+ cells are further genetically modified so as to obtaingenetically modified Pgp+ cells.
 10. The method of claim 9, wherein thegenetically modified Pgp+ cells are selected from the group consistingof T cells and/or NK cells.
 11. The method of claim 1, wherein Pgp+ Tcells are further genetically modified to express at least one chimericantigen receptor, T cell receptor, synthetic immune receptor, chimeric Tcell receptor, or other genetic element so as to obtain geneticallymodified Pgp-positive T cells.
 12. A pharmaceutical composition,comprising: (a) the Pgp+ cells according to claim 1 or geneticallymodified Pgp+ cells thereof; and (b) at least one pharmaceuticallyacceptable carrier.
 13. The pharmaceutical composition of claim 12, foruse in treating cancer, infection or immune disorders.
 14. A method fortreating cancer, infection or immune disorders in a subject, comprising:administering a therapeutically effective amount of the pharmaceuticalcomposition of claim 12 to the subject so as to treat cancer, infectionor immune disorders.
 15. A method for isolating Pgp-negative (Pgp−) Tcells, NK cells or mononuclear cells suitable for adoptive cell transfertherapy, comprising: (a) obtaining a sample comprising T cells, Nk cellsor mononuclear cells; (b) optionally enriching the sample for T cells,NK cells or mononuclear cells; and (c) depleting Pgp-positive (Pgp+) Tcells, NK cells or mononuclear cells from the sample, so as to obtain afraction enriched in Pgp⁻ T cells, NK cells or mononuclear cellssuitable for adoptive cell transfer therapy.
 16. The method of claim 15,wherein depleting the Pgp− T cells, NK cells or mononuclear cells fromthe sample comprises exposing the sample to at least (a) one primaryantibody or antibody like moiety specific to p-glycoprotein; or (b) onephototoxic compound that is a substrate of Pgp.
 17. The method of claim15, wherein the depleting of the Pgp+ T cells, NK cells or mononuclearcells from the sample is performed by any one or more methods selectedfrom immunofluorescent methods, immunomagnetic methods, immunoaffinitymethods, photodepletion or combinations thereof.
 18. The method of claim15, wherein the fraction enriched in Pgp− T cells, NK cells ormononuclear cells contains less than a) 50% Pgp+ T cells, NK cells ormononuclear cells; b) 40% Pgp+ T cells, NK cells or mononuclear cells;c) 30% Pgp+ T cells, NK cells or mononuclear cells; d) 20% Pgp+ T cells,NK cells or mononuclear cells; e) 10% Pgp+ T cells, NK cells ormononuclear cells; f) 5% Pgp+ T cells, NK cells or mononuclear cells; orf) 1% Pgp+ T cells, NK cells or mononuclear cells.
 19. A pharmaceuticalcomposition, comprising: (a) an amount of the fraction enriched in Pgp−T cells, NK cells or mononuclear cells according to claim 15; and (b) atleast one pharmaceutically acceptable carrier.
 20. A method for thetreatment of a subject with immunodeficient HIV/AIDS or a subjectundergoing an allogeneic stem cell transplant comprising: administeringa therapeutically effective amount of the pharmaceutical composition ofclaim 19 to the subject